Macrocyclic compound and uses thereof

ABSTRACT

The present invention provides novel Compound (1) having tumor vascular remodeling effect and/or anti-CAF (Cancer Associated Fibroblasts) activity, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable and medical uses thereof.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational PCT application, PCT/US2018/025887, filed Apr. 3, 2018,which claims priority under 35 U.S.C. § 119(e) to U.S. Provisionalpatent application Ser. No. 62/482,030, filed Apr. 5, 2017, U.S. Ser.No. 62/526,677, filed Jun. 29, 2017, and U.S. Ser. No. 62/586,416, filedNov. 15, 2017, and under 35 U.S.C. § 120 to U.S. patent application Ser.No. 15/814,105, filed Nov. 15, 2017; each of which is incorporatedherein by reference.

TECHNICAL FIELD

The present invention provides a novel macrocyclic compound having tumorvascular remodeling effects and anti-CAF (Cancer Associated Fibroblast)activity. The compound can be used for treating cancer or inhibitingtumor growth in a subject.

BACKGROUND

Halichondrins, such as Halichondrin B, are anticancer agents originallyisolated from the marine sponge Halichondria okadai (See, e.g., D.Uemura et al. “Norhalichondrin A: An Antitumor Polyether Macrolide froma Marine Sponge” J. Am. Chem. Soc., 107, 4796 (1985)), and subsequentlyfound in Axinella sp., Phakellia carteri, and Lissondendryx sp. A totalsynthesis of Halichondrin B was published in 1992 (See, e.g., Y. Kishiet al. “Total Synthesis of Halichondrin B and Norhalichondrin B” J. Am.Chem. Soc., 114, 3162 (1992)). Halichondrin B has demonstrated in vitroinhibition of tubulin polymerization, microtubule assembly, beta5-tubulin crosslinking, GTP and vinblastine binding to tubulin, andtubulin-dependent GTP hydrolysis, and has shown in vitro and in vivoanti-cancer properties (See, e.g., Y. Hirata et al.“Halichondrins-antitumor polyether macrolides from a marine sponge” PureAppl. Chem., 58, 701 (1986); Fodstad et al. “Comparative antitumoractivities of halichondrins and vinblastine against human tumorxenografts” J. of Experimental Therapeutics & Oncology 1996; 1: 119,125).

Eribulin mesylate (Halaven™), which was developed based on HalichondrinB (See, e.g., International Publication No. WO 1999/065894, publishedDec. 23, 1999; International Publication No. WO 2005/118565, publishedDec. 15, 2005; and W. Zheng et al. “Macrocyclic ketone analogues ofhalichondrin B” Bioorganic & Medicinal Chemistry Letters 14, 5551-5554(2004)), is currently in clinical use in many countries for thetreatment of, e.g., metastatic breast cancer and advanced liposarcoma.

Additional patent publications describing Halichondrins include U.S.Pat. No. 5,436,238 to Kishi, et al., issued Jul. 25, 1995; U.S. Pat. No.5,338,865 to Kishi, et al., issued Aug. 16, 1994; and WO 2016/003975filed by Kishi, et al., all of which are assigned to the President andFellows of Harvard College.

See also, e.g., U.S. Pat. Nos. 5,786,492; 8,598,373; 9,206,194;9,469,651; WO/2009/124237A1; WO/1993/017690A1; WO/2012/147900A1; U.S.Pat. Nos. 7,982,060; 8,618,313; 9,303,050; 8,093,410; 8,350,067;8,975,422; 8,987,479; 8,203,010; 8,445,701; 8,884,031; U.S. Pat. No.RE45,324; U.S. Pat. Nos. 8,927,597; 9,382,262; 9,303,039;WO/2009/046308A1; WO/2006/076100A3; WO/2006/076100A2; WO/2015/085193A1;WO/2016/176560A1; U.S. Pat. Nos. 9,278,979; 9,029,573; WO/2011/094339A1;WO/2016/179607A1; WO/2009/064029A1; WO/2013/142999A1; WO/2015/066729A1;WO/2016/038624A1; and WO/2015/000070A1.

Cancer associated fibroblasts (CAFs), which are widely found in avariety of solid tumors, are stromal cells. It is well known that CAFsplay an important role in angiogenesis, invasion, and metastasis. It isreported that there is a close correlation between the amounts of CAFsand clinical prognosis in, for example, invasive breast cancer (See,e.g., M. Yamashita et al. “Role of stromal myofibroblasts in invasivebreast cancer: stromal expression of alpha-smooth muscle actincorrelates with worse clinical outcome” Breast Cancer 19, 170, 2012) andesophageal adenocarcinoma (See, e.g., T. J. Underwood et al.“Cancer-associated fibroblasts predict poor outcome and promoteperiostin-dependent invasion in esophageal adenocarcinoma” Journal ofPathol., 235, 466, 2015). It has also been reported that CAFs correlateto resistance in a variety of tumors such as, for example, breast cancer(See, e.g., P. Farmer et al. “A stroma-related gene signature predictsresistance to neoadjuvant chemotherapy in breast cancer” NatureMedicine., 15(1), 68, 2009), and head and neck cancer (See, e.g., S.Schmitz et al. “Cetuximab promotes epithelial to mesenchymal transitionand cancer associated fibroblasts in patients with head and neck cancer”Oncotarget, 6 (33), 34288, 2015; Y. Matsuoka et al. “The tumor stromalfeatures are associated with resistance to 5-FU-based chemoradiotherapyand a poor prognosis in patients with oral squamous cell carcinoma”APMIS 123(3), 205, 2015).

It has thus been observed that tumor vascular remodeling effects andanti-CAF activity result in the improvement of the cancermicroenvironment, which assists tumor treatment. Blood vessels areessential for the growth of tumors. Reconstructed blood vessels intumors can deliver anti-cancer agents to the tumors, in addition toachieving alleviation of hypoxia. It is reported that eribulin-inducedremodeling of abnormal tumor vasculature leads to a more functionalmicroenvironment that may reduce the aggressiveness of tumors due toelimination of inner tumor hypoxia. Because abnormal tumormicroenvironments enhance both drug resistance and metastasis, theapparent ability of eribulin to reverse these aggressive characteristicsmay contribute to its clinical benefits (See, e.g., Y. Funahashi et al.“Eribulin mesylate reduces tumor microenvironment abnormality byvascular remodeling in preclinical human breast cancer models” CancerSci. 105 (2014), 1334-1342). Anti-cancer drugs having tumor vascularremodeling effects and anti-CAF activities have not been reported as oftoday.

Despite the progress made, additional compounds are needed to progressresearch and medical care of tumors and cancer.

SUMMARY OF THE INVENTION

The present invention relates to a macrocyclic compound (e.g., Compound(1)) having tumor vascular remodeling effects and anti-CAF activity, andpharmaceutically acceptable salts thereof, and isotopically labeledderivatives thereof, and pharmaceutical compositions thereof.

The invention also includes methods of using Compound (1) for treatingcancer, methods for reversibly or irreversibly inhibiting mitosis in acell, and methods for inhibiting tumor growth in vitro, in vivo, or in asubject. In another aspect, the present invention provides kitscomprising Compound (1), or a pharmaceutically acceptable salt thereof,or a pharmaceutical composition thereof.

In one aspect, the invention features a compound which is Compound (1):

and pharmaceutically acceptable salts thereof; and isotopically labeledderivatives thereof.

In one aspect, the invention provides pharmaceutical compositionscomprising Compound (1), or a pharmaceutically acceptable salt orisotopically labeled derivative thereof. The pharmaceutical compositionsmay comprise one or more pharmaceutically acceptable excipients orcarriers. The pharmaceutical compositions may further comprise one ormore additional therapeutic agents in combination, alternation, or otherkind of synchronized therapy, to achieve the desired goal of treatment.

The invention also features methods of making Compound (1) or itsintermediates. The synthetic intermediates are also provided herein aspart of the invention.

It has been discovered that Compound (1) has an advantageous effect ontumor vascular remodeling and has anti-CAF activity, as demonstrated inthe Figures and Examples. Accordingly, the Compound (1) has potentialuse in the treatment of cancer (e.g., squamous cell carcinoma of thehead and neck (SCCHN), breast cancer, esophageal cancer, uterine cancer,ovarian cancer, colorectal cancer, endometrial cancer, gastric cancer,small bowel cancer, bladder cancer, sarcomas, rare cancers).

In another aspect, the present invention provides methods for inhibitingany tumor growth or cancer that will respond to a compound with tumorvascular remodeling effects and/or anti-CAF activity, in a subject,typically a human, with Compound (1), or a pharmaceutically acceptablesalt, or isotopically labeled derivative thereof.

Compound (1), or a pharmaceutically acceptable salt, or isotopicallylabeled derivative thereof, or a composition thereof, may beadministered in combination with any other active agent that providesbeneficial results for the patient. In certain embodiments, Compound (1)is used in combination with an antibody (e.g., a monoclonal antibody).In one embodiment, Compound (1) is used in combination, alternation, orother synchronized therapy with an immunotherapy, such as an anti-EGFR(epidermal growth factor receptor) antibody, an anti-HER2 (humanepidermal growth factor receptor) antibody, an anti-PD-1 antibody, or ananti-PD-L1 antibody, as described in more detail below.

For example, a method is provided to treat squamous cell carcinoma ofthe head and neck (SCCHN) in a subject, typically a human, in needthereof comprising administering to the subject an effective amount ofCompound (1), or a pharmaceutically acceptable salt, or isotopicallylabeled derivative thereof, or a composition thereof, in combinationwith an anti-EGFR (epidermal growth factor receptor) monoclonal antibody(mAb) therapy. In certain embodiments, the anti-EGFR (epidermal growthfactor receptor) mAb is cetuximab.

As another example, a method to treat breast cancer in a subject,typically a human, in need thereof comprising administering to saidsubject an effective amount of Compound (1), or a pharmaceuticallyacceptable salt, or isotopically labeled derivative thereof, or acomposition thereof, in combination with an HER2 (human epidermal growthfactor receptor) mAb therapy. In certain embodiments, the HER2 (humanepidermal growth factor receptor) mAb is trastuzumab. In otherembodiments, the Compound (1) may be used to treat breast cancer incombination with traditional chemotherapy such as adriamycin,cyclophosphamide, taxol, etc., or an anti-estrogen such as a selectiveestrogen modulator (SERM), a selective estrogen degrader (SERD), apartial or total estrogen inhibitor (such as fulvestrant) or a CDK 4/6inhibitor such as palbociclib (Pfizer).

Another aspect of the present invention provides Compound (1), or apharmaceutically acceptable salt, or isotopically labeled derivativethereof, which may be in the form of a hydrate, solvate, polymorph, or acomposition thereof, in a kit, which may be a dosage form package. Thekits described herein may include a single dose or multiple doses of thecompound or pharmaceutical composition thereof. A kit of the inventionmay include instructions for using the provided therapeutic dosage forms(e.g., instructions for using the compound or pharmaceutical compositionincluded in the kit).

The present invention thus includes at least the following features:

-   -   (i) Compound (1), or its pharmaceutically acceptable salt or        isotopically labeled derivative, which may be optionally be in        the form of a hydrate, solvate, or polymorph;    -   (ii) A method for treatment that includes administering an        effective amount to a subject such as a human of Compound (1),        or its pharmaceutically acceptable salt or isotopically labeled        derivative, which may be optionally be in the form of a hydrate,        solvate or polymorph, to treat head and neck cancer (e.g.,        squamous cell carcinoma of the head and neck (SCCHN), adenoid        cystic carcinoma), breast cancer (e.g., HER2-negative breast        cancer, triple negative breast cancer), esophageal cancer (e.g.,        esophageal adenocarcinoma), uterine cancer (e.g., uterine        sarcoma), ovarian cancer, colorectal cancer, sarcoma (e.g.,        synovial sarcoma, angiosarcoma, soft tissue sarcoma,        fibrosarcoma, uterine sarcoma), bladder cancer (e.g., urothelial        cancer), gastric cancer, small bowel cancer (e.g., small bowel        adenocarcinoma), endometrial cancer, or a rare cancer;    -   (iii) A method for treatment that includes administering an        effective amount to a subject such as a human of Compound (1),        or its pharmaceutically acceptable salt or isotopically labeled        derivative, which may be optionally be in the form of a hydrate,        solvate or polymorph, for use in treating a medical disorder        such as a cancer or tumor that responds to vascular remodeling        effects and/or anti-CAF activity;    -   (iv) Compound (1), or its pharmaceutically acceptable salt or        isotopically labeled derivative, which may be optionally be in        the form of a hydrate, solvate or polymorph, for use to treat        squamous cell carcinoma of the head and neck (SCCHN), breast        cancer, esophageal cancer, uterine cancer, ovarian cancer,        colorectal cancer, sarcoma, bladder cancer, gastric cancer,        small bowel cancer, endometrial cancer, or a rare cancer;    -   (v) Compound (1), or its pharmaceutically acceptable salt or        isotopically labeled derivative, which may be optionally be in        the form of a hydrate, solvate or polymorph, for use in treating        a medical disorder such as a cancer or tumor that responds to        vascular remodeling effects and/or anti-CAF activity;    -   (vi) A deuterated derivative of Compound (1);    -   (vii) A process for manufacturing a medicament intended for the        therapeutic use for treating or preventing disorders such as a        cancer or tumor that responds to vascular remodeling effects        and/or anti-CAF activity, characterized in that Compound (1), or        its pharmaceutically acceptable salt or isotopically labeled        derivative, which may be optionally be in the form of a hydrate,        solvate or polymorph described above, or an embodiment of the        active compound, is used in the manufacture;    -   (viii) Compound (1), or its pharmaceutically acceptable salt or        isotopically labeled derivative, in substantially pure form        (e.g., at least 90 or 95%);    -   (ix) A pharmaceutically acceptable composition of Compound (1),        or its pharmaceutically acceptable salt or isotopically labeled        derivative, which may be optionally be in the form of a hydrate,        solvate or polymorph, in a pharmaceutically acceptable carrier        or excipient;    -   (x) A pharmaceutically acceptable dosage form of Compound (1),        or its pharmaceutically acceptable salt or isotopically labeled        derivative, which may be optionally be in the form of a hydrate,        solvate or polymorph, optionally in a pharmaceutically        acceptable carrier or excipient;    -   (xi) Compound (1), or its pharmaceutically acceptable salt or        isotopically labeled derivative, to treat a disorder described        herein whereby it acts through a mechanism other than vascular        remodeling effects and/or anti-CAF activity of action; and    -   (xii) Methods for the manufacture of the compounds described        herein, and intermediates in the synthesis.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, provide non-limitingexamples of the invention.

FIG. 1 shows antitumor effects of Compound (1) in FaDu subcutaneousxenograft model (head and neck cancer) in mice as monotherapy asdescribed in Pharmacological Test Example 4.

FIG. 2 shows antitumor activity of Compound (1) against OSC-19subcutaneous xenograft model (head and neck cancer) in mice asmonotherapy as described in Pharmacological Test Example 5.

FIG. 3 shows antitumor activity of Compound (1) against HCC-1806subcutaneous xenograft (breast cancer) model in mice as monotherapy asdescribed in Pharmacological Test Example 6.

FIG. 4 shows antitumor effects of Compound (1) in FaDu subcutaneousxenograft model in combination with cetuximab in mice as described inPharmacological Test Example 7.

FIG. 5 shows antitumor activity of Compound (1) in KPL-4 subcutaneousxenograft model (breast cancer) in combination with trastuzumab in miceas described in Pharmacological Test Example 8.

FIG. 6A-6B show anti-tumor effect of Compound (1) in HSC-2 orthotopictransplantation mouse model. FIG. 6A. Nude mice were implanted withluciferase-transduced HSC-2 (1×10⁶ cells/spot) in tongue. The amount ofluciferase-transduced HSC-2 was analyzed using In Vivo Imaging System(IVIS). Data show the bioluminescence levels in tongue in each mouse.FIG. 6B. Representative bioluminescence image of 16 mice. CDDP, CTX,CDDP+CTX were used for as comparators, which are currently used intreatment of SCCHN cancer patient treatment. CDDP=cisplatin,CTX=cetuximab.

FIG. 7A-7B show survival advantage of Compound (1) in combination withcetuximab in HSC-2 orthotopic transplantation mouse model. FIG. 7A. Nudemice were implanted with luciferase-transduced HSC-2 (1×10⁶ cells/spot)in tongue. Data show the survival curve until Day 100 after treatment ofdrugs (n=16). *P<0.0001 versus Compound (1) or CTX alone (Log-rank(Mantel-Cox) test). FIG. 7B. The amount of luciferase-transduced HSC-2was analyzed using In Vivo Imaging System (IVIS). Bioluminescence imagesof 10 survived mice of Compound (1)+CTX combination group on Day 100.RBW=relative body weight. CDDP=cisplatin, CTX=cetuximab.

FIG. 8A-8B show anti-tumor effect of Compound (1) in combination withradiation therapy in FaDu mouse xenograft model. FIG. 8A. Nude mice weresubcutaneously implanted with luciferase-transduced FaDu (5×10⁶cells/spot) in the right thighs. Thirteen days after the inoculation,mice were randomly assigned (n=6), and intravenously injected withCompound (1) at 90 μg/kg on Day 1 and Day 8 with or without RT of 18 Gyon Day 4 and Day 11. The amount of luciferase-transduced FaDu wasanalyzed using In Vivo Imaging System (IVIS). Data show the meanrelative bioluminescence level to Day 1 and SEM (n=6). SEM=standarderror of the mean. *P<0.05 versus non-treated on Day 29 (unpairedt-test). FIG. 8B. Representative bioluminescence images of 6 mice eachgroup on Day 29. RT=radiation therapy.

FIG. 9 shows anti-tumor activities of Compound (1) in combination withanti-mPD-1 antibody. CT26 s.c. syngeneic mouse model (colon carcinoma)was treated with Compound (1) and anti-mPD-1 antibody in Q7D scheduleand twice a week schedule, respectively, for 3 weeks. Results showmeans±SEM of tumor volumes (mm³) (n=8).

FIG. 10A shows a cell-free tubulin polymerization assay. Compound (1)has inhibitory activity on tubulin polymerization. FIG. 10B shows amicrotubule dynamics assay. Compound (1) also has inhibitory activity onmicrotubule dynamics.

FIG. 11 shows that Compound (1) is a potent antiproliferative agent inesophageal cancer (OE21, OE33, and TE-8) and uterine cancer (MES-SA,MES-SA/Dx5-Rx1) cell lines.

FIG. 12 shows that Compound (1) has potent anti-tumor activity insubcutaneous xenograft models of breast and ovarian cancer (KPL-4 andCOLO-704, respectively) as a monotherapy.

FIG. 13 shows the effect of Compound (1) on tumor microenvironments. Asshown, Compound (1) increases microvessel density. *P<0.05, **P<0.01,****P<0.0001 versus non-treat (Dunnett multiple comparison test).

FIG. 14 shows the effect of Compound (1) on tumor microenvironments. Asshown, Compound (1) reduces α-SMA positive CAFs.

FIG. 15 shows that Compound (1) decreases ECM proteins from CAFs in FaDusubcutaneous xenograft model. FaDu xenograft tumors were collected onDay 6 after single administration of Compound (1) 180 μg/kg+cetuximab onDay 1.

FIG. 16 shows that Compound (1) exhibits a dose-dependent combinationaleffect with cetuximab in a FaDu subcutaneous xenograft model. Singledose, n=6. Compound (1) and cetuximab (CTX) were administered on Day 1in the FaDu xenograft model.

FIG. 17 shows antitumor effects in the soft tissue sarcoma xenograftmodels in mice as monotherapy. MES-SA (human uterine sarcoma), HT-1080(human fibrosarcoma), and CTG-2041 (human angiosarcoma) are shown.

FIG. 18 shows antitumor effects in endometrial cancer xenograft modelsin mice as monotherapy. HEC-108 and AN3CA (endometrial cancer) areshown.

DEFINITIONS

As used herein, the term “salt” refers to any and all salts, andencompasses pharmaceutically acceptable salts. The term“pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response, and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al. describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 1977, 66, 1-19, incorporated herein by reference.Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids, such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, andperchloric acid or with organic acids, such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid, or malonic acidor by using other methods known in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium, and N⁺(C₁₋₄ alkyl)₄ ⁻ salts.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate, and aryl sulfonate. Compound (1) is alsoprovided, and can be administered, as a free base.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers”. Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers”.

The terms “composition” and “formulation” are used interchangeably.

A “subject” to which administration is contemplated refers to a human(i.e., male or female of any age group, e.g., pediatric subject (e.g.,infant, child, or adolescent) or adult subject (e.g., young adult,middle-aged adult, or senior adult)) or non-human animal. In certainembodiments, the non-human animal is a mammal (e.g., primate (e.g.,cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g.,cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g.,commercially relevant bird, such as chicken, duck, goose, or turkey)).In certain embodiments, the non-human animal is a fish, reptile, oramphibian. The non-human animal may be a male or female at any stage ofdevelopment. The non-human animal may be a transgenic animal orgenetically engineered animal. The term “patient” refers to a humansubject in need of treatment of a disease.

The term “administer,” “administering,” or “administration” refers toimplanting, absorbing, ingesting, injecting, inhaling, or otherwiseintroducing a compound described herein, or a composition thereof, in oron a subject.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, delaying the onset of, or inhibiting the progress of adisease described herein. In some embodiments, treatment may beadministered after one or more signs or symptoms of the disease havedeveloped or have been observed. In other embodiments, treatment may beadministered in the absence of signs or symptoms of the disease. Forexample, treatment may be administered to a susceptible subject prior tothe onset of symptoms. Treatment may also be continued after symptomshave resolved, for example, to delay or prevent recurrence.

An “effective amount” of a compound described herein refers to an amountsufficient to elicit the desired biological response. An effectiveamount of a compound described herein may vary depending on such factorsas the desired biological endpoint, the pharmacokinetics of thecompound, the condition being treated, the mode of administration, andthe age and health of the subject. In certain embodiments, an effectiveamount is a therapeutically effective amount. Alternatively, in aseparate method or use, the invention may be used, where indicated andeffective, as a prophylactic treatment. In certain embodiments, aneffective amount is the amount of a compound described herein in asingle dose. In certain embodiments, an effective amount is the combinedamounts of a compound described herein in multiple doses.

A “therapeutically effective amount” of a compound described herein isan amount sufficient to provide a therapeutic benefit in the treatmentof a condition or to delay or minimize one or more symptoms associatedwith the condition. A therapeutically effective amount of a compoundmeans an amount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms, signs,or causes of the condition, and/or enhances the therapeutic efficacy ofanother therapeutic agent. In certain embodiments, a therapeuticallyeffective amount is an amount sufficient for treating in any disease orcondition described.

As used herein, “inhibition”, “inhibiting”, “inhibit” and “inhibitor”,and the like, refer to the ability of a compound to reduce, slow, halt,or prevent the activity of a biological process (e.g., tumor growth). Incertain embodiments, the inhibition is about 45% to 50%. In certainembodiments, the inhibition is about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9%, or100%.

The terms “neoplasm” and “tumor” are used herein interchangeably andrefer to an abnormal mass of tissue wherein the growth of the masssurpasses and is not coordinated with the growth of a normal tissue. Aneoplasm or tumor may be “benign” or “malignant,” depending on thefollowing characteristics: degree of cellular differentiation (includingmorphology and functionality), rate of growth, local invasion, andmetastasis. A “benign neoplasm” is generally well differentiated, hascharacteristically slower growth than a malignant neoplasm, and remainslocalized to the site of origin. In addition, a benign neoplasm does nothave the capacity to infiltrate, invade, or metastasize to distantsites. In contrast, a “malignant neoplasm” is generally poorlydifferentiated (anaplasia) and has characteristically rapid growthaccompanied by progressive infiltration, invasion, and destruction ofthe surrounding tissue. Furthermore, a malignant neoplasm generally hasthe capacity to metastasize to distant sites. The term “metastasis,”“metastatic,” or “metastasize” refers to the spread or migration ofcancerous cells from a primary or original tumor to another organ ortissue and is typically identifiable by the presence of a “secondarytumor” or “secondary cell mass” of the tissue type of the primary ororiginal tumor and not of that of the organ or tissue in which thesecondary (metastatic) tumor is located.

The term “cancer” refers to a class of diseases characterized by thedevelopment of abnormal cells that proliferate uncontrollably and havethe ability to infiltrate and destroy normal body tissues.

The term “rare cancer” refers to cancers that occur in a relativelysmall number of patients. Rare cancers include, but are not limited to,sarcomas (e.g., soft tissue sarcoma, liposarcoma, uterine sarcoma,leiomyosarcoma, myxofibrosarcoma, osteosarcoma, angiosarcoma, Ewing'ssarcoma, synovial sarcoma, rhabdomyosarcoma), malignant lymphomas,thymic cancer (e.g., thymomas), mesothelioma, gastrointestinal stromaltumors (GISTs), neuroendocrine cancer, eye cancer, brain tumors, bonesoft tissue tumors, skin cancer, and germ cell tumors.

The term “anti-cancer agent” refers to any therapeutic agent that isuseful for treating cancer in a subject (e.g., inhibiting cancer ortumor growth in a subject). Anti-cancer agents encompass biotherapeuticanti-cancer agents as well as chemotherapeutic agents.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention is described in detail below with reference toembodiments and the like of the present invention. The inventionprovides compounds (e.g., Compound (1)), and pharmaceutically acceptablesalts or isotopically labeled derivatives thereof, and pharmaceuticalcompositions thereof. The invention also provides methods of inhibitingtumor growth and/or treating cancer in a subject comprisingadministering an effective amount to the subject of a compound orcomposition provided herein. The compound or composition may beadministered as a monotherapy or in combination with another therapy, asdescribed herein. In yet another aspect, the present invention providesmethods of preparing Compound (1), and synthetic intermediates useful tothat end.

The invention includes a compound of the structure:

or its pharmaceutically acceptable salt or isotopically labeledderivative, which may be optionally be in the form of a hydrate, solvateor polymorph, optionally in a pharmaceutically acceptable carrier orexcipient.

Compound (1) may exist as a crystal polymorph, and the compound of thepresent invention may be in any of single crystal forms or a mixture oftwo or more crystal forms. Compound (1) can be in an amorphous form, orcan be an anhydride or a solvate, such as a hydrate.

The present invention includes isotopically labeled derivatives ofCompound (1) and pharmaceutically acceptable salts thereof. Theisotopically labeled compound is equivalent to Compound (1) except thatone or more of atom(s) are replaced by atom(s) having an atomic mass ora mass number different from those usually found in nature. Examples ofan isotope that can be incorporated into the compound of the presentinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, fluorine, iodine, bromine and chlorine, such as ²H, ³H, ¹¹C,¹³C, ¹⁴C, ¹⁸F, ³⁵S, ¹²³I, and ¹²⁵I.

The isotopically labeled compound, such as a compound into which aradioactive isotope of, for example, ³H and/or ¹⁴C is incorporated, isuseful for a tissue distribution assay for a medicine and/or a matrix.The isotopes ³H and ¹⁴C are regarded to be useful because these isotopescan be easily prepared and detected. The isotopes ¹¹C and ¹⁸F are usefulin PET (positron emission tomography). The isotope ¹²⁵I is regarded tobe useful in SPECT (single photon emission computed tomography), and canbe useful in brain imaging. Replacement by a heavier isotope such as ²Hcauses, because of its higher metabolic stability, some advantages, in atreatment, of, for example, extension of half-life in vivo or reductionof a necessary dose, and therefore, is regarded useful under givencircumstances. The isotopically labeled compound can be similarlyprepared by using a readily available isotopically labeled reagentinstead of a non-isotopically labeled reagent and by performingprocesses disclosed in schemes and/or examples described below.

Compound (1) can be used as a chemical probe for capturing a targetprotein of a biologically active low molecular weight compound.Specifically, the compound of the present invention can be transformedinto an affinity chromatography probe, a photoaffinity probe or the likeby introducing a labeling group, a linker or the like into a moietyother than a structural moiety indispensable to activity expression ofthe compound by a method described in J. Mass Spectrum. Soc. Jpn. Vol.51, No. 5, 2003, p. 492-498, WO2007/139149, or the like.

Examples of the labeling group, the linker or the like used in such achemical probe include groups belonging to the following groups (1) to(5). (1) Protein labeling groups such as photoaffinity labeling groups(such as a benzoyl group, a benzophenone group, an azide group, acarbonyl azide group, a diaziridine group, an enone group, a diazo groupand a nitro group), and chemical affinity groups (such as a ketone groupin which an alpha carbon atom is substituted by a halogen atom, acarbamoyl group, an ester group, an alkylthio group, a Michael acceptorof α,β-unsaturated ketone, ester, or the like, and an oxirane group);(2) cleavable linkers such as S—S, O—Si—O, a monosaccharide (such as aglucose group or a galactose group) and a disaccharide (such aslactose), and oligopeptide linkers that can be cleaved by an enzymereaction; (3) fishing tag groups such as biotin and a 3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacene-3-yl)propionylgroup; (4) radioactive labeling groups such as ¹²⁵I, ³²P, ³H and ¹⁴C;fluorescence labeling groups such as fluorescein, rhodamine, dansyl,umbelliferone, 7-nitrofurazanyl, and a3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacene-3-yl)propionyl group; chemiluminescent groups such as luciferin and luminol;and markers capable of detecting heavy metal ions such as lanthanoidmetal ions and radium ions; and (5) groups to be bonded to a solid phasecarrier such as glass beads, a glass bed, a microliter plate, agarosebeads, an agarose bed, polystyrene beads, a polystyrene bed, nylon beadsand a nylon bed.

A probe prepared by introducing, into the compound of the presentinvention, a labeling group or the like selected from theabove-described groups (1) to (5) by the method described in any of theaforementioned literatures or the like can be used as a chemical probefor identifying a marker protein useful for research of a novelpotential drug target.

Examples of a “salt” used herein include salts with inorganic acids,salts with organic acids, and salts with acidic amino acids, and inparticular, pharmaceutically acceptable salts are preferred. Besides, asalt of the compound of the present invention embraces an anhydride of apharmaceutically acceptable salt thereof and a solvate, such as ahydrate, of the pharmaceutically acceptable salt. Preferable examples ofa salt with an inorganic acid include salts with hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and preferable examples of a salt with an organic acid includesalts with acetic acid, succinic acid, famaric acid, maleic acid,tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and the like. Preferable examples of a salt withan acidic amino acid include salts with aspartic acid and glutamic acidand the like.

In the case where the Compound (1) according to the present invention isobtained as a salt of the Compound (1) or a hydrate of the Compound (1),the salt and the hydrate can be converted to a free body of the Compound(1) by a conventional method.

Pharmaceutical Compositions, Kits, and Administration

The present invention provides pharmaceutical compositions comprisingCompound (1), or a pharmaceutically acceptable salt or isotopicallylabeled derivative thereof, and a pharmaceutically acceptable excipient.In certain embodiments, the compound described herein, orpharmaceutically acceptable salt or isotopically labeled derivativethereof, is provided in an effective amount in the pharmaceuticalcomposition (e.g., a therapeutically effective amount).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include bringing Compound (1) (i.e., the “active ingredient”)into association with a carrier or excipient, and/or one or more otheraccessory ingredients, and then, if necessary and/or desirable, shaping,and/or packaging the product into a desired single- or multi-dose unit.A pharmaceutical composition of the invention could be preparedaccording to the known method such as a method described in the generalrules for preparations of the Japanese Pharmacopoeia, 16^(th) edition,the United States Pharmacopoeia, and the European Pharmacopoeia, 9^(th)edition. A pharmaceutical composition of the invention could beadministered to patients appropriately depending on the dosage form.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.A “unit dose” is a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient which would be administered to a subject and/or a convenientfraction of such a dosage, such as one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition described herein will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.The composition may comprise between 0.1% and 100% (w/w) activeingredient.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

The compound provided herein are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositionsdescribed herein will be decided by a physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular subject or organism will depend upon a varietyof factors including the disease being treated and the severity of thedisorder; the activity of the specific active ingredient employed; thespecific composition employed; the age, body weight, general health,sex, and diet of the subject; the time of administration, route ofadministration, and rate of excretion of the specific active ingredientemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific active ingredient employed; and likefactors well known in the medical arts.

The compound of the present invention (Compound (1)) and compositionsthereof provided herein can be administered by any route, includingenteral (e.g., oral), parenteral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, subcutaneous,intraventricular, transdermal, interdermal, rectal, intravaginal,intraperitoneal, topical (as by powders, ointments, creams, and/ordrops), mucosal, nasal, bucal, sublingual; by intratrachealinstillation, bronchial instillation, and/or inhalation; and/or as anoral spray, nasal spray, and/or aerosol. Specifically contemplatedroutes are oral administration, intravenous administration (e.g.,systemic intravenous injection), regional administration via bloodand/or lymph supply, and/or direct administration to an affected site.In general, the most appropriate route of administration will dependupon a variety of factors including the nature of the agent (e.g., itsstability in the environment of the gastrointestinal tract), and/or thecondition of the subject (e.g., whether the subject is able to tolerateoral administration).

The exact amount of Compound (1) required to achieve an effective amountwill vary from subject to subject, depending, for example, on species,age, and general condition of a subject, severity of the side effects ordisorder, identity of the particular compound, mode of administration,and the like. An effective amount may be included in a single dose(e.g., single oral dose) or multiple doses (e.g., multiple oral doses).In certain embodiments, when multiple doses are administered to asubject or applied to a tissue or cell, any two doses of the multipledoses include different or substantially the same amounts of a compounddescribed herein. In certain embodiments, when multiple doses areadministered to a subject or applied to a tissue or cell, the frequencyof administering the multiple doses to the subject or applying themultiple doses to the tissue or cell may be, in non-limiting examples,three doses a day, two doses a day, one dose a day, one dose every otherday, one dose every third day, one dose every week, one dose every twoweeks, one dose every three weeks, or one dose every four weeks, or evenslow dose controlled delivery over a selected period of time using adrug delivery device. In certain embodiments, the frequency ofadministering the multiple doses to the subject or applying the multipledoses to the tissue or cell is one dose per day. In certain embodiments,the frequency of administering the multiple doses to the subject orapplying the multiple doses to the tissue or cell is two doses per day.In certain embodiments, the frequency of administering the multipledoses to the subject or applying the multiple doses to the tissue orcell is three doses per day. In certain embodiments, when multiple dosesare administered to a subject or applied to a tissue or cell, theduration between the first dose and last dose of the multiple doses isabout or at least one day, two days, four days, one week, two weeks,three weeks, one month, two months, three months, four months, sixmonths, nine months, one year, two years, three years, four years, fiveyears, seven years, ten years, fifteen years, twenty years, or thelifetime of the subject, tissue, or cell. In certain embodiments, theduration between the first dose and last dose of the multiple doses isabout or at least three months, six months, or one year. In certainembodiments, the duration between the first dose and last dose of themultiple doses is the lifetime of the subject, tissue, or cell. Incertain embodiments, a dose (e.g., a single dose, or any dose ofmultiple doses) described herein includes independently between 0.001mg/kg and 0.01 mg/kg, between 0.01 mg/kg and 0.1 mg/kg, or between 0.1mg/kg and 1 mg/kg, inclusive, of Compound (1). Examples are dosage formswith at least about 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 10, 5, 20, 25, or 50 mg of active compound, or its salt, in adosage form.

Dose ranges as described herein provide guidance for the administrationof provided pharmaceutical compositions to an adult. The amount to beadministered to, for example, a child or an adolescent can be determinedby a medical practitioner or person skilled in the art and can be loweror the same as that administered to an adult.

Also encompassed by the disclosure are kits (e.g., pharmaceuticalpacks). The kits provided may comprise a pharmaceutical composition orCompound (1) and a container (e.g., a vial, ampule, bottle, syringe,and/or dispenser package, or other suitable container). In someembodiments, provided kits may optionally further include a secondcontainer comprising a pharmaceutical excipient for dilution orsuspension of a pharmaceutical composition or Compound (1). In someembodiments, the pharmaceutical composition or Compound (1) provided inthe first container and the second container are combined to form oneunit dosage form. A kit described herein may include one or moreadditional pharmaceutical agents described herein as a separatecomposition.

Methods of Treatment and Uses

As shown herein, Compound (1) has significant tumor vascular remodelingeffects and anti-CAF activity, and therefore, it has potential use forthe treatment of cancer and/or the inhibition of tumor growth.

Provided herein is a method of treating cancer in a subject, the methodcomprising administering to the subject an effective amount of aCompound (1), or a pharmaceutically acceptable salt or isotopicallylabeled derivative thereof, or a pharmaceutical composition thereof. Thepresent invention also provides Compound (1), or a pharmaceuticallyacceptable salt or isotopically labeled derivative thereof, or apharmaceutical composition thereof, for use in treating cancer in asubject. The present invention also provides the use of Compound (1), ora pharmaceutically acceptable salt or isotopically labeled derivativethereof, or a pharmaceutical composition thereof, for the manufacture ofa medicament for the treating cancer.

Also provided herein is a method of inhibiting tumor growth in asubject, the method comprising administering to the subject Compound(1), or a pharmaceutically acceptable salt or isotopically labeledderivative thereof, or a pharmaceutical composition thereof. Alsoprovided herein is Compound (1), or a pharmaceutically acceptable saltor isotopically labeled derivative thereof, or a pharmaceuticalcomposition thereof, for use in inhibiting tumor growth in a subject.The present invention also provides the use of Compound (1), or apharmaceutically acceptable salt or isotopically labeled derivativethereof, or a pharmaceutical composition thereof, for the manufacture ofa medicament for inhibiting tumor growth.

In certain embodiments of the methods and uses provided herein, thecancer is head and neck cancer, breast cancer, esophageal cancer,uterine cancer, ovarian cancer, colorectal cancer, endometrial cancer,gastric cancer, small bowel cancer, bladder cancer, or a sarcoma

In certain embodiments of the methods and uses provided herein, thecancer is head and neck cancer (e.g., head and neck squamous cellcarcinoma, oral cancer, throat cancer, salivary gland cancer, tonguecancer, adenoid cystic carcinoma). In certain embodiments, the cancer issquamous cell carcinoma of the head and neck (SCCHN). In certainembodiments, the cancer is adenoid cystic carcinoma. In certainembodiments, the cancer is breast cancer (e.g., HER2-positive breastcancer, triple negative breast cancer). In certain embodiments, thecancer is HER2-positive breast cancer. In certain embodiments, thecancer is triple negative breast cancer. In certain embodiments, thecancer is colorectal cancer (e.g., colon carcinoma). In certainembodiments, the cancer is colon carcinoma. In certain embodiments, thecancer is esophageal cancer (e.g., esophageal adenocarcinoma). Incertain embodiments, the cancer is esophageal adenocarcinoma. In certainembodiments, the cancer is uterine cancer (e.g., uterine sarcoma). Incertain embodiments, the cancer is uterine sarcoma. In certainembodiments, the cancer is ovarian cancer. In certain embodiments, thecancer is a sarcoma (e.g., uterine sarcoma, fibrosarcoma, angiosarcoma,synovial sarcoma, soft tissue carcinoma). In certain embodiments, thecancer is fibrosarcoma. In certain embodiments, the cancer isangiosarcoma. In certain embodiments, the cancer is synovial sarcoma. Incertain embodiments, the cancer is soft tissue carcinoma. In certainembodiments, the cancer is gastric cancer. In certain embodiments, thecancer is bowel cancer (e.g., small bowel cancer, small boweladenocarcinoma). In certain embodiments, the cancer is small bowelcancer. In certain embodiments, the cancer is small boweladenocarcinoma. In certain embodiments, the cancer is bladder cancer(e.g., urothelial cancer). In certain embodiments, the cancer isurothelial cancer. In certain embodiments, the cancer is endometrialcancer. In certain embodiments, the cancer is a rare cancer.

Combination Therapy

Besides administration as monotherapy, Compound (1) can be administeredin combination with other therapeutic agents or treatment modalities. Incertain embodiments, the additional therapeutic agent is an antibody. Incertain embodiments, the additional therapeutic agent is a monoclonalantibody. The compound of the present invention can be administered incombination with another therapeutic agent, such as anti-EGFR therapy,anti-HER2 therapy, anti-PD-1 therapy, anti-PD-L1 therapy, or irradiationtherapy.

In certain embodiments, Compound (1), or a pharmaceutically acceptablesalt or isotopically labeled derivative thereof, or a pharmaceuticalcomposition thereof, is administered in combination with an anti-EGFRtherapy (e.g., anti-EGFR monoclonal antibody (mAb), such as cetuximab).In certain embodiments, the anti-EGFR therapy is an anti-EGFR antibody.For example, provided herein is a method of treating squamous cellcarcinoma of the head and neck (SCCHN) in a subject comprisingadministering to said subject Compound (1), or a pharmaceuticallyacceptable salt or isotopically labeled derivative thereof, or apharmaceutical composition thereof, in combination with an anti-EGFR(epidermal growth factor receptor) mAb therapy. In certain embodiments,the anti-EGFR mAb is cetuximab (CTX).

In certain embodiments, Compound (1), or a pharmaceutically acceptablesalt or isotopically labeled derivative thereof, or a pharmaceuticalcomposition thereof, is administered in combination with an anti-HER2therapy (e.g., anti-HER2 monoclonal antibody (mAb) such as trastuzumab).In certain embodiments, the anti-HER2 therapy is an anti-HER2 antibody.For example, provided herein is a method of treating breast cancer in asubject in need thereof comprising administering to said subjectCompound (1), or a pharmaceutically acceptable salt or isotopicallylabeled derivative thereof, or a composition thereof, in combinationwith an HER2 (human epidermal growth factor receptor) mAb therapy. Incertain embodiments, the anti-HER2 mAb is trastuzumab.

In certain embodiments, Compound (1), or a pharmaceutically acceptablesalt or isotopically labeled derivative thereof, or a pharmaceuticalcomposition thereof, is administered in combination with an anti-PD-1 oranti-PD-L1 therapy (e.g., anti-PD-1 or anti-PD-L1 monoclonal antibody).In certain embodiments, the anti-PD-1 or anti-PD-L1 therapy is anantibody. For example, provided herein is a method of treatingcolorectal cancer in a subject in need thereof comprising administeringto said subject Compound (1), or a pharmaceutically acceptable salt orisotopically labeled derivative thereof, or a composition thereof, incombination with an anti-PD-1 or anti-PD-L1 therapy (e.g., mAb therapy).

In certain embodiments, Compound (1), or a pharmaceutically acceptablesalt or isotopically labeled derivative thereof, or a pharmaceuticalcomposition thereof, is used in combination with radiation therapy (RT).In certain embodiments, the compound is administered in combination withsurgery.

EXAMPLES

Synthesis of Compound (1)

General Procedures and Methods

The compound according to the present invention can be produced by themethods described in Examples below. However, these examples are onlyfor illustrative purposes, and the compound according to the presentinvention is not limited to the specific examples mentioned below in anyway.

In the Examples, unless specifically mentioned otherwise, the silica gelfor purification by using silica gel column chromatography was Hi-Flash™Column (Silica Gel, 30 μm 60 Å or 40 μm 60 Å, Yamazen Corporation), thesilica gel for purification by using NH silica gel column chromatographywas Chromatorex NH silica gel (Fuji Silysia Chemical LTD). Analyticalthin layer chromatography (TLC) was performed with TLC silica gel 60F₂₅₄, layer thickness 0.25 mm (Merck KGaA) or Chromatorex TLC NH silicagel F₂₅₄, layer thickness 0.25 mm (Fuji Silysia Chemical LTD). TLCplates were visualized by staining with p-anisaldehyde stain,phosphomolybdic acid stain or Hanessian's Stain.

All moisture sensitive reactions were conducted under an inertatmosphere. Reagents and solvents were commercial grade and were used assupplied, unless otherwise noted.

NMR spectra were recorded on a JEOL ECZ500R (500 MHz), JEOL ECZ400S (400MHz), Varian Inova 500 (500 MHz), Varian Mercury 400 (400 MHz) or BrukerAvance (600 MHz) spectrometer. Chemical shifts are reported in parts permillion (ppm). For ¹H NMR spectra (CDCl₃, C₆D₆, and/or CD₃OD), theresidual solvent peak was used as the internal reference (7.27 ppm inCDCl₃; 7.16 ppm in C₆D₆; 3.31 ppm in CD₃OD).

Analytical mass spectra (MS) results were obtained using a WatersAcquity UPLC equipped with a single quadrapole detector (SQ Detector 2)or LTQ Orbitrap XL™ (Thermoscientific).

High performance liquid chromatography (HPLC) was carried out withShimadzu LC-10AD on a UV spectrophotometric detector (200 nm, ShimadzuSPD-10A).

The abbreviations used herein are as follows: AIBN:2,2′-azobis(isobutyronitrile); 9-BBN: 9-borabicyclo[3.3.1]nonane;Bu₃SnH: tri-normal-butyltin hydride; (+)-CSA:(1S)-(+)-10-Camphorsulfonic acid; DMAP: 4-dimethylaminopyridine; DCM:dichloromethane; DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DIBAL:diisobutylaluminium hydride; DMF: N, N-dimethylformamide; DMSO: dimethylsulfoxide; Et₃N: triethylamine; EtOAc: ethyl acetate; HF-Pyridine:hydrogen fluoride pyridine; HPLC: high performance liquidchromatography; IPA: isopropyl alcohol; MeCN: acetonitrile; MeOH:methanol; MPM: para-methoxybenzyl; PPh₃: triphenylphosphine; t-BuOH:tertiary-butyl alcohol; tBuLi: tertiary-butyl lithium; TBME: methyltertiary-butyl ether; TBAF: tetrabutylammonium fluoride; TBS:tertiary-butyldimethylsilyl; THF: tetrahydrofuran; TMS: trimethylsilyl;Ts: para-toluenesulfonyl.

The synthetic intermediates disclosed herein are considered part of thepresent invention.

Example 1(4aR,5aS,6R,8aS,9aR)-2,2-di-tert-butyl-6-methyloctahydrofuro[2′,3′:5,6]pyrano[3,2-d][1,3,2]dioxasilin-7-ol

Under a nitrogen atmosphere, to the solution of Compound A-1:

(4aR,5aS,6R,8aS,9aR)-2,2-di-tert-butyl-6-methylhexahydrofuro[2′,3′:5,6]pyrano[3,2-d][1,3,2]dioxasilin-7(8aH)-one(A-1 18.5 g, 54.0 mmol) obtained by the method written in OrganicLetters (2009), 11(2), 409-412 (CAS No; 1095280-04-8) in toluene (275mL) at −78° C., DIBAL (70.2 mL, 70.2 mmol, 1.0 M toluene solution) wasadded over 30 min. Then the reaction mixture was stirred at −78° C.After 90 min, the reaction was quenched with MeOH (4.37 mL) carefully at−78° C., then removed the cooling bath. Saturated potassium sodiumtartrate tetrahydrate solution (300 mL) was added to the reactionmixture, continued stirring for 2 hr at room temperature. The reactionmixture was poured into a separatory funnel, then the layers wereseparated. The aqueous layer was extracted with EtOAc (300 mL). Thecombined organic extracts were washed with brine (300 mL), dried overNa₂SO₄, filtered, concentrated under reduced pressure. The crude lactolwas used for the next reaction without purification.

Example 2(4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butylhexahydropyrano[3,2-d][1,3,2]dioxasilin-7-ol(Compound A-2)

Under a nitrogen atmosphere, to the suspension ofmethyltriphenylphosphonium bromide (73.30 g, 205.2 mmol) in THF (200mL), potassium tert-butoxide (17.27 g, 153.9 mmol) was added at −5° C.over 10 min, and then stirred for 60 min at −5° C. Solution of the crudelactol described in Example 1 in THF (40 mL) was transferred to thereaction mixture at −5° C. over 10 min, then stirred at −5° C. for 1 hr,at room temperature for 1 hr. The reaction mixture was quenched withice-water (400 mL), then diluted with TBME (400 mL) and then the layerswere separated. The aqueous layer was extracted with TBME (400 mL). Thecombined organic extracts were washed with brine (400 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas suspended with Heptane/EtOAc=1/1 (100 mL). The resulting suspensionwas filtered, rinsed with Heptane/EtOAc=1/1 (100 mL) to removetriphenylphosphine derived material. Then filtrate was concentratedunder reduced pressure. Flash chromatography of the residue on silicagel (400 g, Silica Gel 60, spherical, 40-50 μm, Kanto Chemical) using 0%to 20% EtOAc/Heptane gave the title compound (Compound A-2, 16.7 g, 90%yield).

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.03 (d, J=6.8 Hz, 3H) 1.05 (s, 9H)1.07 (s, 9H) 1.75 (dt, J=14.5, 3.0 Hz, 1H) 2.37 (dt, J=14.5, 2.9 Hz, 1H)2.65-2.76 (m, 1H) 3.03 (dd, J=9.8, 1.0 Hz, 1H) 3.31 (m, 1H) 3.69 (d,J=15.0 Hz, 1H) 3.75-3.79 (m, 1H) 4.16-4.31 (m, 2H) 4.41 (t, J=2.9 Hz,1H) 4.95-5.09 (m, 2H) 6.02 (ddd, J=17.3, 10.5, 6.3 Hz, 1H).

Example 3(4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)hexahydropyrano[3,2-d][1,3,2]dioxasiline (Compound A-3)

Under a nitrogen atmosphere, to a solution of Compound A-2:

(4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butylhexahydropyrano[3,2-d][1,3,2]dioxasilin-7-ol(9.85 g, 28.8 mmol) described in Example 2 in DCM (150 mL) at 0° C. wereadded 2,6-lutidine (6.68 mL, 57.5 mmol) and tert-butyldimethylsilyltrifluoromethanesulfonate (9.25 mL, 40.3 mmol). The reaction mixture wasstirred at 0° C. for 30 min, then at room temperature for 2 hr. Thereaction mixture was diluted with diethyl ether. The organic layer waswashed with 0.5 N HCl aq, sat. NaHCO₃ aq and then brine. The combinedorganic layers were dried over MgSO₄, filtered (small amount of SiO₂)and concentrated under reduced pressure. Flash chromatography of theresidue on silica gel using 0% to 15% EtOAc/Heptane gave the titlecompound (Compound A-3, 12.0 g, 91% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.10 (s, 3H) 0.19 (s, 3H) 0.91 (s,9H) 0.96 (d, J=6.3 Hz, 3H) 1.02 (s, 9H) 1.06 (s, 9H) 1.73 (dt, J=15.0,4.0 Hz, 1H) 2.26 (dt, J=15.0, 2.5 Hz, 1H) 2.66-2.74 (m, 1H) 2.95 (dd,J=9.5, 2.2 Hz, 1H) 3.17 (m, 1H) 3.81-3.84 (m, 1H) 4.12-4.22 (m, 2H) 4.24(t, J=2.7 Hz, 1H) 4.93-5.06 (m, 2H) 6.08 (ddd, J=17.3, 10.5, 6.3 Hz,1H).

Example 4(2R,3R,5S,6S)-6-((S)-but-3-en-2-yl)-5-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3-ol (Compound A-4)

Under a nitrogen atmosphere, to a solution of Compound A-3:

(4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)hexahydropyrano[3,2-d][1,3,2]dioxasiline (12 g, 26.3 mmol) described inExample 3 in MeCN (120 mL) and DCM (40 mL) at −10° C. was addedpre-mixed solution of HF-Pyridine (4.0 mL) and pyridine (20 mL) in 20 mLof MeCN. The reaction mixture was stirred at −10° C. for 15 min, then atroom temperature for 1 hr. The reaction mixture was quenched withsat.NaHCO₃ aq at 0° C. and diluted with DCM, then the layers wereseparated. The aqueous layer was extracted with DCM. The combinedorganic extracts were washed with brine. The combined organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressure.Flash chromatography of the residue on silica gel using 15% to 60%EtOAc/Heptane gave the title compound (Compound A-4, 8.4 g, Quant.yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.13 (s, 3H) 0.19 (s, 3H) 0.94 (s,9H) 0.96 (d, J=6.8 Hz, 3H) 1.72 (dt, J=14.6, 2.9 Hz, 1H) 2.15 (dd,J=9.8, 2.4 Hz, 1H) 2.23 (dt, J=14.6, 2.9 Hz, 1H) 2.55-2.65 (m, 1H) 3.03(d, J=9.8 Hz, 1H) 3.41-3.46 (m, 1H) 3.49 (d, J=11.7 Hz, 1H) 3.62-3.72(m, 2H) 3.92 (ddd, J=11.7, 8.3, 2.4 Hz, 1H) 4.02 (t, J=2.7 Hz, 1H)5.01-5.12 (m, 2H) 5.93 (ddd, J=17.4, 10.4, 7.3 Hz, 1H).

Example 5(((2S,3S,5R,6R)-2-((S)-but-3-en-2-yl)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,5-diyl)bis(oxy))bis(tert-butyldimethylsilane)(Compound A-5)

Under a nitrogen atmosphere, to a solution of Compound A-4:

(2R,3R,5S,6S)-6-((S)-but-3-en-2-yl)-5-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3-ol (997 mg, 3.15 mmol) described in Example 4 in DCM(10 mL) at 5° C. was added 2,6-lutidine (1.83 mL, 15.8 mmol) andtert-butyldimethylsilyl trifluoromethanesulfonate (2.17 mL, 9.45 mmol).The reaction mixture was stirred at room temperature for 5 hr. Thereaction mixture was diluted with diethyl ether and quenched with sat.NaHCO₃ aq, then the layers were separated. The combined organic extractswere successively washed with 0.5 N HCl aq, sat.NaHCO₃ aq, and thenbrine. The organic layer was dried over MgSO₄, filtered and concentratedunder reduced pressure. Flash chromatography of the residue on silicagel using 0% to 5% EtOAc/Heptane (containing 1% Et₃N) gave the titlecompound (Compound A-5, 1.69 g, 98% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.02-0.08 (m, 15H) 0.11 (s, 3H)0.89 (s, 9H) 0.90-0.92 (m, 18H) 0.94 (d, J=6.8 Hz, 3H) 1.82 (dt, J=14.9,4.8 Hz, 1H) 2.00 (dt, J=14.9, 2.9 Hz, 1H) 2.62-2.72 (m, 1H) 2.93 (dd,J=9.3, 2.0 Hz, 1H) 3.27-3.34 (m, 1H) 3.66-3.79 (m, 3H) 3.83-3.87 (m, 1H)4.91-5.07 (m, 2H) 6.11 (ddd, J=17.3, 10.7, 6.1 Hz, 1H).

Example 6(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butan-1-ol (Compound A-6)

To a solution of Compound A-5:

(((2S,3S,5R,6R)-2-((S)-but-3-en-2-yl)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,5-diyl)bis(oxy))bis(tert-butyldimethylsilane)(1.32 g, 2.42 mmol) described in Example 5 in THF (10 mL) at 0° C. wasadded 9-BBN (9.69 mL, 0.5 M THF solution, 4.84 mmol). The reactionmixture was stirred at 0° C. for 1 hr and at room temperature for 1.5hr. 3.0 M NaOH aq (3 mL, 9.00 mmol) and hydrogen peroxide (35% in water,3 mL) were added to the reaction mixture at 0° C. The reaction mixturewas stirred at 0° C. for 30 min, then at room temperature for 1 hr. Thereaction mixture was quenched with sat. Na₂SO₃ aq and then the layerswere separated. The aqueous layer was extracted with EtOAc (3 times).The combined organic extracts were washed with brine, dried over Na₂SO₄,filtered and concentrated under reduced pressure. Flash chromatographyof the residue on silica gel using 0% to 20% EtOAc/Heptane gave thetitle compound (Compound A-6, 1.36 g, 100% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.03 (s, 3H) 0.05-0.08 (m, 12H)0.10 (s, 3H) 0.88 (d, J=6.8 Hz, 3H) 0.89-0.93 (m, 27H) 1.55-1.65 (m, 1H)1.82 (dt, J=15.4, 4.4 Hz, 1H) 1.87-1.96 (m, 1H) 1.97-2.03 (m, 1H)2.17-2.26 (m, 1H) 2.67 (dd, J=7.8, 3.9 Hz, 1H) 2.98-3.10 (m, 1H)3.34-3.40 (m, 1H) 3.59-3.86 (m, 6H) ESI-MS (m/z): 563.64 [M+H]⁺, 585.62[M+Na]⁺

Example 7(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butanal (Compound A-7)

Under a nitrogen atmosphere, to a solution of Compound A-6:

(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butan-1-ol (1100 mg, 1.954 mmol)described in Example 6 in DCM (30 mL) at 5° C. were added NaHCO₃ (41.0mg, 0.49 mmol) and Dess-Martin periodinane (1077 mg, 2.54 mmol). Thereaction mixture was stirred at room temperature. After 3 hr, thereaction mixture was diluted with DCM and quenched with sat.NaHCO₃ aqand sat. Na₂SO₃ aq, then the layers were separated. The aqueous layerwas extracted with DCM. The combined organic extracts were washed withbrine, dried over MgSO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 0% to25% EtOAc/Heptane gave the title compound (Compound A-7, 950 mg, 87%yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.00 (s, 3H) 0.03-0.08 (m, 12H)0.11 (s, 3H) 0.88 (s, 9H) 0.91-0.92 (m, 21H) 1.82 (dt, J=15.0, 4.5 Hz,1H) 2.01 (dt, J=15.0, 2.5 Hz, 1H) 2.28 (ddd, J=16.0, 7.3, 2.4 Hz, 1H)2.53-2.58 (m, 1H) 2.74 (ddd, J=16.0, 5.5, 2.0 Hz, 1H) 2.94 (dd, J=9.0,1.7 Hz, 1H) 3.29 (td, J=5.9, 2.0 Hz, 1H) 3.68 (d, J=5.9 Hz, 2H)3.75-3.82 (m, 1H) 3.82-3.90 (m, 1H) 9.73 (t, J=2.4 Hz, 1H).

Example 8(2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)pent-4-yn-1-yl4-methylbenzenesulfonate (Compound B-2)

Under a nitrogen atmosphere, to the solution of Compound B-1:

(2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)pent-4-yn-1-ol(11.08 g, 36.15 mmol) obtained by the method written in WO 9317690A1/U.S. Pat. No. 5,436,238 A (CAS No; 157323-41-6) in DCM (330 mL), Et₃N(12.6 mL, 90.4 mmol) and p-toluenesulfonyl chloride (8.27 g, 43.4 mmol)were added at room temperature. The reaction mixture was stirred at roomtemperature overnight. The mixture was washed with sat.NaHCO₃ and brine,dried over MgSO₄, filtered, then concentrated under reduced pressure.Flash chromatography of the residue on silica gel (Silica Gel 60,spherical, 40-50 μm, Kanto Chemical) using 0% to 10% EtOAc/Heptane gavethe title compound (Compound B-2, 17.7 g, 93% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.17 (s, 9H) 1.02 (d, J=6.8 Hz, 3H)2.10-2.18 (m, 1H) 2.44 (s, 3H) 3.82 (s, 3H) 3.99 (d, J=6.8 Hz, 1H)4.04-4.07 (m, 2H) 4.33 (d, J=11.2 Hz, 1H) 4.66 (d, J=11.2 Hz, 1H) 6.87(d, J=8.3 Hz, 2H) 7.21 (d, J=8.3 Hz, 2H) 7.33 (d, J=8.8 Hz, 2H) 7.77 (d,J=8.8 Hz, 2H).

Example 9((3S,4R)-5-iodo-3-((4-methoxybenzyl)oxy)-4-methylpent-1-yn-1-yl)trimethylsilane(Compound B-3)

Under a nitrogen atmosphere, to the solution of Compound B-2:

(2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)pent-4-yn-1-yl4-methylbenzenesulfonate (17.7 g, 38.4 mmol) described in Example 8 inDMF (360 mL), NaI (7.49 g, 50.0 mmol) was added at room temperature. Thereaction mixture was stirred at 80° C. for 2 hr. Another 2.0 g of NaIwas added to the reaction mixture. The reaction was stirred for 1.5 hrat 80° C., then cooled to room temperature. The mixture was diluted withdiethyl ether, washed with water and brine, dried over MgSO₄, filteredand concentrated under reduced pressure. Flash chromatography of theresidue on silica gel (Silica Gel 60, spherical, 40-50 μm, KantoChemical) using 10% to 20% EtOAc/Heptane gave the title compound(Compound B-3, 14.3 g, 89% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.21 (s, 9H) 1.10 (d, J=6.8 Hz, 3H)1.74-1.84 (m, 1H) 3.30-3.37 (m, 2H) 3.82 (s, 3H) 3.96 (d, J=7.3 Hz, 1H)4.44 (d, J=11.2 Hz, 1H) 4.73 (d, J=11.2 Hz, 1H) 6.89 (d, J=8.8 Hz, 2H)7.30 (d, J=8.8 Hz, 2H).

Example 10(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(trimethylsilyl)non-8-yn-4-ol(Compound C-1)

Under an argon atmosphere, to a solution of Compound B-3:

((3S,4R)-5-iodo-3-((4-methoxybenzyl)oxy)-4-methylpent-1-yn-1-yl)trimethylsilane(1408 mg, 3.382 mmol) described in Example 9 in diethyl ether (25 mL) at−78° C. was added tert-butyllithium (1.61M in pentane, 4.11 mL, 6.62mmol). The reaction mixture was stirred at −78° C. for 45 min. CompoundA-7:(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butanal (825 mg, 1.47 mmol) described inExample 7 in 5.0 mL of diethyl ether was added to the reaction mixtureat −78° C. The reaction mixture was stirred at −78° C. for 60 min. Thereaction mixture was quenched with sat.NH₄Cl aq. Organic layer waswashed with brine, dried over Na₂SO₄, then concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 0% to25% EtOAc/Heptane gave the title compound (Compound C-1, 1167 mg, 93%yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.00-0.12 (m, 21H) 0.15-0.24 (m,6H) 0.82-0.96 (m, 30H) 1.03 (d, J=6.3 Hz, 3H) 1.38-1.55 (m, 1H)1.68-1.99 (m, 4H) 2.10-2.30 (m, 2H) 2.76-2.87 (m, 1H) 3.15 (d, J=9.75Hz, 1H) 3.33-3.38 (m, 1H) 3.56-4.02 (m, 9H) 4.37-4.50 (m, 1H) 4.64-4.78(m, 1H) 6.83-6.88 (m, 2H) 7.23-7.35 (m, 2H).

Example 11(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(tributylstannyl)non-8-en-4-ol(Compound C-3)

To a solution of Compound C-1:

(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(trimethylsilyl)non-8-yn-4-ol(1165 mg, 1.37 mmol) described in Example 10 in MeOH (20 mL) at 20° C.was added K₂CO₃ (189 mg, 1.37 mmol). The reaction mixture was stirred at20° C. for 2 hr. The reaction mixture was diluted with EtOAc andquenched with sat.NH₄Cl aq, then the layers were separated. The aqueouslayer was extracted with EtOAc. The combined organic extracts werewashed with brine, dried over MgSO₄, filtered and concentrated underreduced pressure. Flash chromatography of the residue on silica gelusing 0% to 15% EtOAc/Heptane gave Compound C-2:(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methylnon-8-yn-4-ol(1050 mg, 98% yield). ESI-MS (m/z): 801.50 [M+Na]⁺

Under a nitrogen atmosphere, to a solution of Compound C-2:

(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methylnon-8-yn-4-ol(780 mg, 1.00 mmol) obtained above in toluene (15 mL) at 20° C. wereadded tri-n-butyltin hydride (2.5 mL, 9.36 mmol) and2,2′-azobis(isobutyronitrile) (82 mg, 0.50 mmol). The reaction mixturewas stirred at 90° C. for 15 min. The reaction mixture was concentratedunder reduced pressure. Flash chromatography of the residue on silicagel using 0% to 15% EtOAc/Heptane gave the title compound (Compound C-3,970 mg, 91% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.02-0.13 (m, 18H) 0.84-0.96 (m,48H) 1.22-1.37 (m, 6H) 1.47-1.56 (m, 7H) 1.72-1.90 (m, 3H) 1.95-2.03 (m,1H) 2.11-2.28 (m, 2H) 2.82-2.86 (m, 1H) 3.08-3.15 (m, 1H) 3.33-3.40 (m,1H) 3.43-3.53 (m, 1H) 3.58-3.87 (m, 8H) 4.25-4.31 (m, 1H) 4.49-4.54 (m,1H) 5.83 (dd, J=19.3, 7.6 Hz, 1H) 6.05-6.13 (m, 1H) 6.83-6.90 (m, 2H)7.24 (d, J=8.8 Hz, 2H).

Example 12(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(Compound C-4)

Under a nitrogen atmosphere, to a solution of Compound C-3:

(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(tributylstannyl)non-8-en-4-ol(970 mg, 0.91 mmol) described in Example 11 in 30 mL of DCM at 5° C. wasadded iodine (242 mg, 0.95 mmol) in DCM (6 mL) until it maintained theiodine color. The reaction mixture was quenched with sat.Na₂SO₃ aq andthe layers were separated. The aqueous layer was extracted with DCM. Thecombined organic extracts were washed with brine. The combined organiclayers were dried over Na₂SO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 0% to25% EtOAc/Heptane gave(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-ol(768 mg, 93% yield).

Under a nitrogen atmosphere, to a solution of

(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-ol(768 mg, 0.85 mmol) obtained above in DCM (25 mL) at room temperaturewas added NaHCO₃ (17.8 mg, 0.21 mmol) and Dess-Martin periodinane (485mg, 1.14 mmol). The reaction mixture was stirred at room temperature for4 hr. The reaction mixture was diluted with DCM and quenched withsat.NaHCO₃ aq and sat.Na₂SO₃ aq, and then the layers were separated. Theaqueous layer was extracted with DCM. The combined organic extracts werewashed with brine, dried over MgSO₄, filtered and concentrated underreduced pressure. Flash chromatography of the residue on silica gelusing 0% to 20% EtOAc/Heptane gave the title compound (Compound C-4, 776mg, Quant. yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.00 (s, 3H) 0.03-0.07 (m, 12H)0.10 (s, 3H) 0.81 (d, J=6.3 Hz, 3H) 0.84 (d, J=6.3 Hz, 3H) 0.89 (s, 9H)0.91 (s, 9H) 0.92 (s, 9H) 1.80 (dt, J=15.0, 4.5 Hz, 1H) 1.99 (dt,J=15.0, 2.5 Hz, 1H) 2.17 (dd, J=16.6, 10.2 Hz, 1H) 2.20-2.29 (m, 2H)2.43-2.48 (m, 1H) 2.54 (d, J=12.7 Hz, 1H) 2.87 (dd, J=9.0, 1.7 Hz, 1H)2.99 (dd, J=16.6, 2.9 Hz, 1H) 3.27 (td, J=5.8, 2.4 Hz, 1H) 3.50-3.56 (m,1H) 3.66-3.74 (m, 2H) 3.75-3.78 (m, 1H) 3.80 (s, 3H) 3.81-3.85 (m, 1H)4.26 (d, J=11.7 Hz, 1H) 4.50 (d, J=11.7 Hz, 1H) 6.26 (d, J=14.6 Hz, 1H)6.42 (dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.3 Hz, 2H) 7.21 (d, J=8.3 Hz,2H). ESI-MS (m/z): 927.39 [M+Na]⁺

Example 13(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(Compound C-5)

To a solution of Compound C-4:

(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(600 mg, 0.66 mmol) described in Example 12 in THF (5.0 mL), IPA (5.0mL) and t-BuOH (5.0 mL) at 4° C. was added (1S)-(+)-10-Camphorsulfonicacid (154 mg, 0.66 mmol). The reaction mixture was stirred at 4° C. for20 hr. The reaction mixture was diluted with EtOAc and quenched withsat.NaHCO₃ aq, then the layers were separated. The aqueous layer wasextracted with EtOAc. The combined organic extracts were washed withbrine, dried over MgSO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 0% to35% EtOAc/Heptane gave the title compound (Compound C-5, 500 mg, 95%yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.01 (s, 3H) 0.04 (s, 3H) 0.07 (s,3H) 0.11 (s, 3H) 0.86-0.91 (m, 15H) 0.93 (s, 9H) 1.83 (dt, J=14.9, 4.8Hz, 1H) 1.93-2.00 (dt, J=14.9, 4.8 Hz, 1H) 2.19-2.26 (m, 1H) 2.29 (dd,J=14.9, 5.6 Hz, 1H) 2.39 (dd, J=16.6, 8.3 Hz, 1H) 2.44-2.66 (m, 4H) 2.91(dd, J=9.5, 1.7 Hz, 1H) 3.36-3.41 (m, 1H) 3.48 (td, J=11.3, 2.7 Hz, 1H)3.59 (t, J=7.1 Hz, 1H) 3.74-3.78 (m, 2H) 3.80 (s, 3H) 3.85 (m, 1H) 4.25(d, J=11.2 Hz, 1H) 4.46 (d, J=11.2 Hz, 1H) 6.28 (d, J=14.6 Hz, 1H) 6.43(dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.8 Hz, 2H) 7.21 (d, J=8.8 Hz, 2H).ESI-MS (m/z): 813.30 [M+Na]⁺

Example 14((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl4-methylbenzenesulfonate (Compound C-6)

Under a nitrogen atmosphere, to a solution of Compound C-5:

(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(500 mg, 0.63 mmol) described in Example 13 in DCM (10 mL) at 5° C. wereadded pyridine (2.54 mL, 31.6 mmol), p-toluenesulfonyl chloride (723 mg,3.79 mmol) and 4-dimethylaminopyridine (77 mg, 0.63 mmol). The reactionmixture was stirred at room temperature for 24 hr. p-Toluenesulfonylchloride (150 mg, 0.79 mmol) was added to the reaction mixture at roomtemperature. Then, the reaction mixture was stirred at room temperaturefor 8 hr. The reaction mixture was diluted with DCM and quenched withsat.NaHCO₃ aq, then the layers were separated. The aqueous layer wasextracted with DCM. The combined organic extracts were washed withbrine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 0% to25% EtOAc/Heptane gave the title compound (Compound C-6, 560 mg, 94%yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.01 (s, 3H) 0.04 (s, 3H) 0.04 (s,3H) 0.08 (s, 3H) 0.81 (d, J=6.8 Hz, 3H) 0.83 (s, 9H) 0.86 (d, J=6.8 Hz,3H) 0.89 (s, 9H) 1.81 (dt, J=14.9, 4.5 Hz, 1H) 1.91-1.96 (m, 1H)2.15-2.32 (m, 3H) 2.36-2.42 (m, 1H) 2.43 (s, 3H) 2.57 (d, J=12.7 Hz, 1H)2.77 (dd, J=16.6, 3.4 Hz, 1H) 2.87 (dd, J=9.0, 1.7 Hz, 1H) 3.53-3.58 (m,2H) 3.70-3.75 (m, 1H) 3.80-3.85 (m, 1H) 3.81 (s, 3H) 4.06 (dd, J=10.0,5.0 Hz, 1H) 4.08-4.16 (m, 1H) 4.28 (d, J=11.2 Hz, 1H) 4.51 (d, J=11.2Hz, 1H) 6.30 (d, J=14.6 Hz, 1H) 6.45 (dd, J=14.6, 7.8 Hz, 1H) 6.88 (d,J=8.8 Hz, 2H) 7.24 (d, J=8.8 Hz, 2H) 7.31 (d, J=8.3 Hz, 2H) 7.76 (d,J=8.3 Hz, 2H).

Example 15(2S,6S,7S,E)-2-((2S,3S,5R,6R)-6-(azidomethyl)-3,5-bis((tert-butyldimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(Compound C-7)

Under a nitrogen atmosphere, to a solution of Compound C-6:

((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl4-methylbenzenesulfonate (560 mg, 0.59 mmol) described in Example 14 inDMSO (5.6 mL) at 20° C. was added sodium azide (385 mg, 5.92 mmol). Thereaction mixture was stirred at 85° C. After 2 hr, sodium azide (100 mg,1.54 mmol) was added to the reaction mixture, then the reaction mixturewas stirred at 85° C. for 14 hr. The reaction mixture was diluted withEtOAc and quenched with H₂O, then the layers were separated. The organicextracts were successively washed with water and brine. The combinedorganic layers were dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a crude residue. Flash chromatography of theresidue on silica gel using 0% to 15% EtOAc/Heptane gave the titlecompound (Compound C-7, 298 mg, 62% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.03 (s, 3H) 0.06 (s, 3H) 0.07 (s,3H) 0.10 (s, 3H) 0.84 (d, J=6.8 Hz, 3H) 0.85 (d, J=6.8 Hz, 3H) 0.91 (s,9H) 0.92 (s, 9H) 1.86 (dt, J=15.0, 4.7 Hz, 1H) 1.98 (dt, J=15.0, 2.9 Hz,1H) 2.19-2.32 (m, 3H) 2.41-2.49 (m, 1H) 2.58 (d, J=12.7 Hz, 1H) 2.94(dd, J=16.6, 2.9 Hz, 1H) 2.98 (dd, J=8.8, 2.0 Hz, 1H) 3.02 (dd, J=12.7,2.9 Hz, 1H) 3.47 (dt, J=8.8, 2.7 Hz, 1H) 3.49-3.54 (m, 1H) 3.63 (dd,J=12.7, 8.8 Hz, 1H) 3.69-3.73 (m, 1H) 3.81 (s, 3H) 3.83-3.88 (m, 1H)4.26 (d, J=11.7 Hz, 1H) 4.50 (d, J=11.7 Hz, 1H) 6.26 (d, J=14.6 Hz, 1H)6.42 (dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.8 Hz, 2H) 7.22 (d, J=8.8 Hz,2H).

Example 16(((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl)carbamate(Compound C-8)

To a solution of Compound C-7:

(2S,6S,7S,E)-2-((2S,3S,5R,6R)-6-(azidomethyl)-3,5-bis((tert-butyldimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(298 mg, 0.37 mmol) described in Example 15 in THF (10 mL) and water(1.0 mL) at 20° C. was added triphenylphosphine (1437 mg, 5.478 mmol).The reaction mixture was stirred at 70° C. for 1.5 hr. The reactionmixture was concentrated under reduced pressure to give a crude amine.To a solution of the crude amine obtained above in THF (10 mL) at 5° C.were added Et₃N (0.51 mL, 3.66 mmol) and diallyl dicarbonate (341 mg,1.83 mmol). The reaction mixture was stirred at room temperature for 60min. The reaction mixture was concentrated under reduced pressure. Flashchromatography of the residue on silica gel using 0% to 25%EtOAc/Heptane gave the title compound (Compound C-8, 300 mg, 94% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.05-0.07 (m, 9H) 0.11 (s, 3H) 0.85(d, J=6.3 Hz, 3H) 0.87 (d, J=6.3 Hz, 3H) 0.90 (s, 9H) 0.93 (s, 9H) 1.80(dt, J=15.0, 4.4 Hz, 1H) 1.96 (dt, J=15.0, 2.8 Hz, 1H) 2.16-2.29 (m, 2H)2.32-2.39 (m, 1H) 2.53-2.60 (m, 3H) 2.86 (d, J=7.3 Hz, 1H) 3.04-3.11 (m,1H) 3.30-3.34 (m, 1H) 3.38-3.48 (m, 1H) 3.58 (t, J=7.1 Hz, 1H) 3.70-3.76(m, 1H) 3.80 (s, 3H) 3.81-3.84 (m, 1H) 4.25 (d, J=11.2 Hz, 1H) 4.46 (d,J=11.2 Hz, 1H) 4.53-4.63 (m, 2H) 5.19 (dd, J=10.7, 1.5 Hz, 1H) 5.32 (d,J=17.1 Hz, 1H) 5.47 (d, J=6.8 Hz, 1H) 5.88-5.99 (m, 1H) 6.28 (d, J=14.6Hz, 1H) 6.43 (dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.8 Hz, 2H) 7.21 (d,J=8.8 Hz, 2H). ESI-MS (m/z): 896.34 [M+Na]⁺

Example 17

Under a nitrogen atmosphere (in a glove box), to a solution of CompoundD-2:

(S)—N-(2-(4-isopropyl-4,5-dihydrooxazol-2-yl)-6-methoxyphenyl)methanesulfonamide(155 mg, 0.497 mmol) obtained by the method written in Organic Letters(2002), 4 (25), 4431-4434 (CAS No; 546141-34-8) and1,8-bis(dimethylamino)naphthalene (107 mg, 0.497 mmol) in MeCN (0.75 mL)was added chromium(II) chloride (55.5 mg, 0.452 mmol) and then theresulting mixture was stirred in the glove box at room temperature for 1hr. The resulting green solution was added to a mixture of Compound C-8:allyl(((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl)carbamate(99.0 mg, 0.113 mmol) described in Example 16, Compound D-1 (80.0 mg,0.09 mmol) obtained by the method written in Journal of the AmericanChemical Society (1992), 114 (8), 3162-3164 (CAS No; 157322-23-1),Compound D-3: dichloro(2,9-dimethyl-1,10-phenanthroline)nickel (0.46 mg,1.36 μmol) obtained by the method written in Journal of the AmericanChemical Society (2009), 131(42), 15387-15393 (CAS No; 21361-04-6) andlithium chloride (3.83 mg, 0.09 mmol). The reaction mixture was stirredin the glove box at room temperature for 60 min. The reaction mixturewas then taken out of the glove box, diluted with diethyl ether-EtOAc(5.0 mL-5.0 mL), then Florisil® (1600 mg, 15.94 mmol) (CAS No;1343-88-0) was added to the mixture. Then mixture was stirred at roomtemperature for 30 min. The mixture was filtered (Celite®), washed withEtOAc/Heptane=2/1, then filtrate was concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 3% to55% EtOAc/Heptane gave the title compound (Compound D-4, 140 mg, 95%yield).

Example 18

Under a nitrogen atmosphere, to a solution of Compound D-4 (140 mg, 0.09mmol) described in Example 17 in DCM (5.0 mL) at 5° C. were added NaHCO₃(28.8 mg, 0.34 mmol) and Dess-Martin periodinane (72.7 mg, 0.17 mmol).The reaction mixture was stirred at room temperature for 60 min. Thereaction mixture was diluted with DCM and quenched with sat. NaHCO₃ aqand sat. Na₂SO₃ aq, and then the layers were separated. The aqueouslayer was extracted with DCM. The combined organic extracts were washedwith brine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 2% to60% EtOAc/Heptane gave the title compound (Compound D-5, 120 mg, 86%).

¹H NMR (500 MHz, BENZENE-d6) δ ppm 0.01-0.05 (m, 9H) 0.10-0.12 (m, 6H)0.15 (s, 3H) 0.76 (d, J=6.1 Hz, 3H) 0.96 (s, 9H) 1.02 (s, 9H) 1.04 (s,9H) 0.95-1.10 (m, 7H) 1.20 (d, J=7.3 Hz, 3H) 1.31-1.37 (m, 3H) 1.41 (dd,J=12.8, 4.9 Hz, 1H) 1.40-1.58 (m, 4H) 1.59-1.64 (m, 1H) 1.69-1.89 (m,3H) 1.90-1.99 (m, 2H) 2.02-2.25 (m, 8H) 2.26-2.48 (m, 6H) 2.49-2.70 (m,6H) 2.71-2.84 (m, 2H) 3.00-3.07 (m, 1H) 3.12-3.30 (m, 4H) 3.36 (s, 3H)3.40 (br.s, 1H) 3.44-3.53 (m, 2H) 3.65 (dd, J=6.4, 4.0 Hz, 1H) 3.69-3.84(m, 4H) 3.86-4.03 (m, 4H) 4.07-4.17 (m, 3H) 4.27-4.29 (m, 1H) 4.27 (d,J=11.0 Hz, 1H) 4.48-4.58 (m, 1H) 4.49 (d, J=11.0 Hz, 1H) 4.65-4.70 (m,2H) 4.68 (d, J=5.5 Hz, 1H) 4.74-4.86 (m, 2H) 4.78 (s, 1H) 4.93 (s, 1H)5.05 (d, J=10.4 Hz, 1H) 5.09 (br. s., 1H) 5.19 (br. s., 1H) 5.30 (dd,J=17.1, 1.2 Hz, 1H) 5.82 (d, J=8.0 Hz, 1H) 5.86-5.96 (m, 1H) 6.46 (d,J=15.9 Hz, 1H) 6.84-6.92 (m, 3H) 7.31 (d, J=8.6 Hz, 2H).

Example 19

Imidazole hydrochloride (155 mg, 1.48 mmol) was dissolved in DMF (2.9mL) to give a 0.5 M imidazole hydrochloride solution in DMF. 1.0 mL ofthis solution was mixed with 1.0 mL of TBAF (1.0 M, THF solution) togive a premixed solution of 0.5 M TBAF and 0.25 M imidazolehydrochloride in THF-DMF (1:1). Under a nitrogen atmosphere, to asolution of Compound D-5 (80.0 mg, 0.05 mmol) described in Example 18 inDMF (7.0 mL) at 20° C. were added 0.588 mL of premixed solution of TBAF(0.5 M) and imidazole hydrochloride (0.25 M) in THF-DMF (1:1) preparedabove. The reaction mixture was stirred at room temperature for 14 hr.1.6 g of CaCO₃ and 4.0 g of Dowex® 50WX8 (hydrogen form, 200-400 mesh,SIGMA-ALDRICH) were added to the reaction mixture. The mixture wasstirred at room temperature for 2 hr. Then mixture was diluted withEtOAc, then filtered (Celite®), washed with EtOAc. Filtrate wasconcentrated under reduced pressure to give a crude residue. 1000 mg ofCaCO₃ and 2.25 g of Dowex® 50WX8 were added to the EtOAc (6.0 mL)solution of the crude residue. The mixture was stirred at roomtemperature for 2.5 hr. Then mixture was diluted with EtOAc, filtered(Celite®), washed with EtOAc. Filtrate was concentrated under reducedpressure to give a crude residue (63.0 mg). To a solution of the cruderesidue (63.0 mg) obtained above in DCM (6.0 mL), t-BuOH (0.6 mL) and pH7 Phosphate Buffer (0.6 mL, 1/15 M) at room temperature was added DDQ(111 mg, 0.49 mmol). The reaction mixture was stirred at roomtemperature for 45 min. The reaction mixture was quenched with sat.NaHCO₃ aq, then diluted with DCM and the layers were separated. Theaqueous layer was extracted with DCM (3 times). The combined organicextracts were dried over Na₂SO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on NH silica gel using 10%to 100% EtOAc/Heptane, then 10% MeOH/EtOAc gave a roughly purified titlecompound (Compound D-6, 15.0 mg, 27%).

¹H NMR (500 MHz, METHANOL-d4) δ ppm 0.97 (d, J=7.0 Hz, 3H) 0.97 (d,J=7.0 Hz, 3H) 1.00-1.02 (m, 1H) 1.05 (d, J=7.3 Hz, 3H) 1.09 (d, J=6.3Hz, 3H) 1.31-1.45 (m, 6H) 1.46-1.63 (m, 5H) 1.64-1.75 (m, 3H) 1.80-1.86(m, 2H) 1.87-1.93 (m, 2H) 1.94-2.11 (m, 9H) 2.13-2.27 (m, 8H) 2.33 (d,J=2.4 Hz, 2H) 2.39 (dd, J=13.4, 6.1 Hz, 1H) 2.44 (dd, J=17.6, 2.0 Hz,1H) 2.55 (dd, J=17.6, 9.3 Hz, 1H) 2.75-2.84 (m, 1H) 2.97 (dd, J=9.3, 2.0Hz, 1H) 3.21 (dd, J=6.6, 4.6 Hz, 1H) 3.32 (m, 1H) 3.41-3.46 (m, 1H) 3.57(br. s., 1H) 3.60 (d, J=11.7 Hz, 1H) 3.67-3.74 (m, 2H) 3.78 (br. s., 1H)3.86-3.90 (m, 2H) 3.97 (d, J=2.4 Hz, 1H) 4.02-4.11 (m, 4H) 4.17 (dd,J=6.6, 4.6 Hz, 1H) 4.23 (dd, J=11.5, 2.2 Hz, 1H) 4.29 (br.s, 1H) 4.31(td, J=9.3, 3.9 Hz, 1H) 4.44 (d, J=10.2 Hz, 1H) 4.51 (d, J=5.4 Hz, 2H)4.59 (t, J=4.9 Hz, 1H) 4.61 (dd, J=7.3, 4.9 Hz, 1H) 4.69 (t, J=4.6 Hz,1H) 4.80 (s, 1H) 4.85-4.87 (m, 1H) 5.01 (s, 1H) 5.05 (s, 1H) 5.16 (dd,J=10.7, 1.0 Hz, 1H) 5.28 (dd, J=17.1, 2.0 Hz, 1H) 5.92 (m, 1H). ESI-MS(m/z): 1172.57 [M+Na]⁺

Example 20

Under a nitrogen atmosphere, to a solution of Compound D-6 (15.0 mg,0.013 mmol) described in Example 19, pyrrolidine (10.8 μL, 0.13 mmol) inDCM (2.0 mL) at room temperature was addedtetrakis(triphenylphosphine)palladium(0) (7.53 mg, 6.52 μmol). Thereaction mixture was stirred at room temperature for 30 min. Thereaction mixture was concentrated under reduced pressure. Flashchromatography of the residue on NH silica gel using 50% EtOAc/Heptane,then 0% to 20% MeOH/EtOAc to give a roughly purified product. Obtainedroughly purified product was purified by HPLC to give the title compound(D-7, 7.0 mg, 47%, retention time=13.8 min).

HPLC Conditions:

Column: YMC Pak Pro C18 (20 mm×250 mm)

Detection wavelength: 200 nm

Column temperature: room temperature

Mobile phase: MeCN-Water (0.05% AcOH)

Flow rate: 8 mL/min

Eluate:

MeCN/Water 25% (iso, 2 min), then

MeCN/Water 25% to 60% (Gradient, 20 min)

¹H NMR (500 MHz, METHANOL-d4) δ ppm 0.99 (d, J=6.7 Hz, 3H) 1.00-1.03 (m,1H) 1.04 (d, J=7.3 Hz, 3H) 1.06 (d, J=7.3 Hz, 3H) 1.10 (d, J=6.1 Hz, 3H)1.29-1.63 (m, 10H) 1.65-1.78 (m, 3H) 1.79-1.89 (m, 2H) 1.92-2.12 (m,10H) 1.93 (s, 3H) 2.13-2.36 (m, 9H) 2.41 (dd, J=13.5, 6.1 Hz, 1H) 2.45(dd, J=17.6, 2.2 Hz, 1H) 2.56 (dd, J=17.6, 9.8 Hz, 1H) 2.75-2.84 (m, 1H)2.98 (dd, J=9.8, 1.8 Hz, 1H) 3.12 (dd, J=12.8, 3.7 Hz, 1H) 3.22 (dd,J=6.4, 4.6 Hz, 1H) 3.26 (dd, J=13.2, 7.8 Hz, 1H) 3.39 (d, J=1.8 Hz, 1H)3.61 (d, J=12.8 Hz, 1H) 3.63-3.68 (m, 2H) 3.68-3.76 (m, 2H) 3.81-3.94(m, 3H) 4.00 (d, J=2.5 Hz, 1H) 4.03-4.15 (m, 4H) 4.18 (dd, J=6.4, 4.6Hz, 1H) 4.25 (ddd, J=11.0, 4.3, 1.8 Hz, 1H) 4.27-4.36 (m, 2H) 4.46 (d,J=11.0 Hz, 1H) 4.57-4.65 (m, 2H) 4.70 (t, J=4.6 Hz, 1H) 4.81 (d, J=1.2Hz, 1H) 5.02 (br. s, 1H) 5.06 (d, J=1.8 Hz, 1H). ESI-MS (m/z): 1066.96[M+H]⁺, 1090.19 [M+Na]⁺

Compound (1) (salt free form of Compound D-7): ¹H NMR (600 MHz,METHANOL-d4) δ ppm 0.98 (d, J=7.2 Hz, 3H) 1.00 (d, J=6.8 Hz, 3H) 1.02(m, 1H) 1.05 (d, J=6.8 Hz, 3H) 1.09 (d, J=6.4 Hz, 3H) 1.28-1.45 (m, 5H)1.46-1.59 (m, 4H) 1.57-1.63 (m, 1H) 1.65-1.71 (m, 1H) 1.70-1.75 (m, 2H)1.79-1.86 (m, 2H) 1.91 (dt, J=14.9, 3.1 Hz, 1H) 1.94-2.11 (m, 8H)2.14-2.34 (m, 9H) 2.39 (dd, J=13.2, 6.0 Hz, 1H) 2.44 (dd, J=17.4, 1.9Hz, 1H) 2.56 (dd, J=17.6, 9.6 Hz, 1H) 2.69 (dd, J=13.2, 4.2 Hz, 1H) 2.79(ddq, J=15.9, 7.6, 2.0 Hz, 1H) 2.92 (dd, J=13.2, 8.3 Hz, 1H) 2.97 (dd,J=9.6, 1.7 Hz, 1H) 3.21 (dd, J=6.4, 4.9 Hz, 1H) 3.29 (m, 1H) 3.34 (dd,J=8.3, 4.15 Hz, 1H) 3.58 (br. s., 1H) 3.60 (br. d, J=11.3 Hz, 1H)3.68-3.73 (m, 2H) 3.80 (br. s., 1H) 3.84-3.90 (m, 2H) 3.98 (d, J=2.3 Hz,1H) 4.03-4.13 (m, 4H) 4.17 (dd, J=6.4, 4.9 Hz, 1H) 4.24 (ddd, J=11.3,4.5, 1.5 Hz, 1H) 4.29 (dd, J=4.0, 1.9 Hz, 1H) 4.32 (td, J=10.2, 4.2 Hz,1H) 4.44 (br. d, J=11.0 Hz, 1H) 4.59 (t, J=4.5 Hz, 1H) 4.62 (dd, J=7.4,4.7 Hz, 1H) 4.69 (t, J=4.7 Hz, 1H) 4.80 (br. s., 1H) 4.87 (s, 1H) 5.00(br. s., 1H) 5.05 (br. d, J=1.1 Hz, 1H)

ESI-MS (m/z): 1066.57 [M+H]⁺, 1088.55 [M+Na]⁺

Pharmacological Test Examples

General Information

Natural Halichondrin compounds and modified compounds thereof are knownin the literature (See, e.g., D. Uemura et al. “Norhalichondrin A: AnAntitumor Polyether Macrolide from a Marine Sponge” J. Am. Chem. Soc.,107, 4796 (1985); Marc Litaudon et al. “Antitumor Polyether Macrolides:New and Hemisynthetic Halichondrins from the New Zealand Deep-WaterSponge Lissodendoryx sp.” J. Org. Chem., 1997, 62, 1868-1871). However,most of them are not easily available. For example, Dr. Uemura et. al.isolated 12.5 mg of Halichondrin B, 35.0 mg of Norhalichondrin A and17.2 mg of Homohalichondrin A from as much as 600 kg of Halichondriaokadai Kadota (See, e.g., D. Uemura et al. “Norhalichondrin A: AnAntitumor Polyether Macrolide from a Marine Sponge” J. Am. Chem. Soc.,107, 4796 (1985)). Among natural Halichondrin compounds, Halichondrin Bshows the strongest anti-tumor activities against B-16 melanoma cells invitro and is highly active against L-1210 Leukemia in vivo (See, e.g.,D. Uemura et al. “Norhalichondrin A: An Antitumor Polyether Macrolidefrom a Marine Sponge” J. Am. Chem. Soc., 107, 4796 (1985)). HalichondrinC is also active in various in vivo models but unstable in aqueoussolution in comparison with Halichondrin B. Norhalichondrin B is muchweaker than Halichondrin B not only in vitro but also in vivo See, e.g.,D. Uemura et al. “Norhalichondrin A: An Antitumor Polyether Macrolidefrom a Marine Sponge” J. Am. Chem. Soc., 107, 4796 (1985)). Thefollowing pharmacological tests use Halichondrin B (Hali-B) as referencecompounds as needed.

Pharmacological Test Example 1. FaDu Growth Inhibition Assay

In this assay, the growth inhibitory activity of test compounds in ahuman squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu was measured. FaDu cells were maintained in an RPMI-1640 (Wako PureChemical Industries, Ltd., 187-02021) medium containing 10% fetal bovineserum (FBS: Nichirei, 12D168), and penicillin and streptomycin in a 5%CO₂ incubator (37° C.). To each well of a 96 well plate (Becton,Dickinson and Company, 353219), 75 μL of FaDu cell suspension adjustedto a concentration of 4×10⁴ cells/mL with the culture medium was added,and the cells were incubated overnight in a 5% CO₂ incubator (37° C.).On the next day, 25 μL of Compound (1) or Halichondrin B in three-folddilution series suspended in the culture medium was added to each well,and the resultant was incubated for 3 days in a 5% CO₂ incubator (37°C.). Then, cell viability was determined by CellTiter-Glo® LuminescentCell Viability Assay (Promega) with EnVision 2103 Multilabel Reader(Perkin-Elmer, Wellesley, Mass.). Value of the wells containing cellswithout adding the test compounds was defined as 100% and the value ofthe wells containing no cells was defined as 0%. The concentration ofthe test compound necessary for inhibiting the cell growth by 50% (i.e.,an IC₅₀ value) was calculated, and shown in Table 1.

TABLE 1 FaDu Test compound (IC₅₀ (nM)) Halichondrin B 0.124 Compound (1)0.0714

Pharmacological Test Example 2. MDA-MB231 Growth Inhibition Assay

In this assay, the growth inhibitory activity of test compounds in ahuman breast cancer cell line MDA-MB231 was measured. MDA-MB231 cellswere maintained in Dulbecco's Modified Eagle's medium (Wako PureChemical Industries, Ltd., 044-29765) medium containing 10% fetal bovineserum (FBS: Nichirei, 12D168), and penicillin and streptomycin in a 5%CO₂ incubator (37° C.). To each well of a 96 well plate (Becton,Dickinson and Company, 353219), 75 μL of MDA-MB231 cell suspensionadjusted to a concentration of 4×10⁴ cells/mL with the culture mediumwas added, and the cells were incubated overnight in a 5% CO₂ incubator(37° C.). On the next day, 25 μL of Compound (1) or Halichondrin B inthree-fold dilution series suspended in the culture medium was added toeach well, and the resultant was incubated for 3 days in a 5% CO₂incubator (37° C.). Then, cell viability was determined byCellTiter-Glo® Luminescent Cell Viability Assay (Promega) with EnVision2103 Multilabel Reader (Perkin-Elmer, Wellesley, Mass.). Value of thewells containing cells without adding the test compounds was defined as100% and the value of the wells containing no cells was defined as 0%.The concentration of the test compound necessary for inhibiting the cellgrowth by 50% (i.e., an IC₅₀ value) was calculated, and shown in Table2.

TABLE 2 MDA-MB231 Test compound (IC₅₀ (nM)) Halichondrin B 1.000Compound (1) 0.109

Pharmacological Test Example 3. HCC1954 Growth Inhibition Assay

In this assay, the growth inhibitory activity of test compounds in ahuman breast cancer cell line HCC1954 was measured. HCC1954 cells weremaintained in an RPMI-1640 medium modified to contain 2 mM L-glutamine,10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/Lsodium bicarbonate (ATCC 30-2001) containing 10% fetal bovine serum(FBS: Nichirei, 12D168), and penicillin and streptomycin in a 5% CO₂incubator (37° C.). To each well of a 96 well plate (Becton, Dickinsonand Company, 353219), 75 μL of HCC1954 cell suspension adjusted to aconcentration of 4×10⁴ cells/mL with the culture medium was added, andthe cells were incubated overnight in a 5% CO₂ incubator (37° C.). Onthe next day, 25 μL of Compound (1) or Halichondrin B in three-folddilution series suspended in the culture medium was added to each well,and the resultant was incubated for 3 days in a 5% CO₂ incubator (37°C.). Then, cell viability was determined by CellTiter-Glo® LuminescentCell Viability Assay (Promega) with EnVision 2103 Multilabel Reader(Perkin-Elmer, Wellesley, Mass.). Value of the wells containing cellswithout adding the test compounds was defined as 100% and the value ofthe wells containing no cells was defined as 0%. The concentration ofthe test compound necessary for inhibiting the cell growth by 50% (i.e.,an IC₅₀ value) was calculated, and shown in Table 3.

TABLE 3 HCC1954 Test compound (IC₅₀ (nM)) Halichondrin B 0.154 Compound(1) 0.0668

Pharmacological Test Example 4. Antitumor Effects in FaDu SubcutaneousXenograft Model in Mice as Monotherapy

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin and streptomycin, was adjusted to a concentration of4.8×10⁷ cells/mL with Hanks' Balanced Salt Solution to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 7 weeks of ages(CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles River Laboratories JapanInc.). Nine days after cell inoculation, the shortest diameter and thelongest diameter of a tumor in each mouse were measured by using anelectronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation),so as to calculate the volume of the tumor in accordance with thefollowing calculation formulae:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday) Tumor Regression(%)=(1−minimum RTV)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. Each test compoundwas dissolved in DMSO and a solution was stored at the freezer beforeuse. Immediately before the administration, the stock solution wasdiluted in saline with 100 μM of hydroxypropyl-β-cyclodextrin. Eachevaluation sample was intravenously administered at a maximum tolerabledose (MTD). Incidentally, the experiment was conducted on groups eachconsisting of 4 mice. Tumor regression (%) of each test compound wasshown in Table 4.

TABLE 4 Test compound Dose (mg/kg) Tumor Regression (%) Halichondrin B0.05 0 Compound (1) 0.2 43

Pharmacological Test Example 5. Antitumor Activity Against OSC-19 inSubcutaneous Xenograft Model in Mice as Monotherapy

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineOSC-19, which had been cultured in an DMEM/Ham's F-12 (1:1) mediumcontaining 10% FBS, and penicillin and streptomycin, was adjusted to aconcentration of 1×10⁸ cells/ml with PBS to prepare a cell suspension,and the suspension was mixed with Matrigel™ (BD Bioscience, #366237) ina ratio of 1:1 to prepare a cell suspension in a concentration of 5×10⁷cell/mL. The cell suspension was inoculated in a volume of 100 μL into asubcutaneous part of a right flank of nude mice, 5 weeks of ages(CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Six days after cell inoculation, the shortest diameter and thelongest diameter of a tumor in each mouse were measured by using anelectronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation),so as to calculate the volume of the tumor in accordance with thefollowing calculation formulae:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)Tumor Regression(%)=(1−minimum RTV)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. The experiment wasconducted on groups each consisting of 6 mice. Test compound wasdissolved in saline and intravenously administered at doses from 0.06mg/kg to 0.18 mg/kg once a week for 2 weeks (Q7D×2 schedule). Tumorregression (%) of each test dose is shown in Table 5.

TABLE 5 Test compound Dose (mg/kg) Tumor Regression (%) Compound (1)0.06 59 Compound (1) 0.18 90

Pharmacological Test Example 6. Antitumor Activity Against HCC1806 inSubcutaneous Xenograft Model in Mice as Monotherapy

A human breast cancer cell line HCC1806, which had been cultured in anRPMI-1640 medium containing 10% FBS, and penicillin and streptomycin,was adjusted to a concentration of 1×10⁸ cells/mL with PBS to prepare acell suspension, and the suspension was mixed with Matrigel™ (BDBioscience, #366237) in a ratio of 1:1 to prepare a cell suspension in aconcentration of 5×10⁷ cell/mL. The cell suspension was inoculated in avolume of 100 μL into a subcutaneous part of a right flank of nude mice,5 weeks of ages (CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles RiverLaboratories Japan, Inc.). Twelve days after cell inoculation, theshortest diameter and the longest diameter of a tumor in each mouse weremeasured by using an electronic digital caliper (Digimatic™ caliper,Mitutoyo Corporation), so as to calculate the volume of the tumor inaccordance with the following calculation formulae:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)Tumor Regression(%)=(1−minimum RTV)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. The experiment wasconducted on groups each consisting of 6 mice. Test compound wasdissolved in saline and intravenously administered at 0.18 mg/kg once aweek for 2 weeks (Q7D×2 schedule). Tumor regression (%) for Compound (1)is shown in Table 6.

TABLE 6 Test compound Dose (mg/kg) Tumor Regression (%) Compound (1)0.18 90

Pharmacological Test Example 7. Antitumor Effects in FaDu SubcutaneousXenograft Model in Combination with Cetuximab in Mice

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin and streptomycin, was adjusted to a concentration of5×10⁷ cells/mL with Hanks' Balanced Salt Solution to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 7 weeks of ages(CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles River Laboratories JapanInc.). Ten days after cell inoculation, the shortest diameter and thelongest diameter of a tumor in each mouse were measured by using anelectronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation),so as to calculate the volume of the tumor in accordance with thefollowing calculation formulae:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)Tumor Regression on day 35(%)=(1−RTV on day 35)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. Each test compoundwas dissolved in DMSO and a solution was stored at the freezer beforeuse. Immediately before the administration, the stock solution wasdiluted in saline with 100 μM of hydroxypropyl-β-cyclodextrin. Each testcompound and was intravenously administered at doses from ¼ MTD to ½ MTDin combination with cetuximab (Erbitux, Merck Serono Co., Ltd.).Incidentally, the experiment was conducted on groups each consisting of4 mice. Tumor regression on day 35(%) of each test compound are shown inTable 7.

TABLE 7 Dose Cetuximab Tumor Regression Test compound (mg/kg) (mg/kg) onday 35 (%) — — 20 −242 Halichondrin B 0.0125 20 −38 0.025 20 −2 Compound(1) 0.05 20 38 0.1 20 60

Pharmacological Test Example 8. Antitumor Activity in KPL-4 SubcutaneousXenograft Model in Combination with Trastuzumab in Mice

A human HER-2 positive breast cancer cell line KPL-4, which had beencultured in an RPMI-1640 medium containing 10% FBS, and penicillin andstreptomycin, was adjusted to a concentration of 1×10⁸ cells/mL withHank's Balanced Salt Solution to prepare a cell suspension. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 7 weeks of ages (CAnN.Cg-Foxn1nu/CrlCrlj,female, Charles River Laboratories Japan, Inc.). Sixteen days after thecell inoculation, the shortest diameter and the longest diameter of atumor in each mouse were measured by using an electronic digital caliper(Digimatic™ caliper, Mitutoyo Corporation), so as to calculate thevolume of the tumor in accordance with the following calculationformulae:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)Tumor Regression(%)=(1−minimum RTV)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. The experiment wasconducted on groups each consisting of 6 mice. Each test compound wasdissolved in DMSO and a solution was stored at the freezer before use.Immediately before the administration, the stock solution was diluted insaline. The test compound was intravenously administered at 0.09 mg/kgor 0.18 mg/kg in combination with trastuzumab (Herceptin, Genentech,Inc.). Tumor regression for Compound (1) is shown in Table 8.

TABLE 8 Trastuzumab Tumor Regression Test compound Dose (mg/kg) (mg/kg)(%) — — 10 0 Compound (1) 0.09 — 43 0.09 10 83 0.18 — 87 0.18 10 100

Pharmacological Test Example 9. Effect on CD31-Positive Vessel in theFaDu Subcutaneous Model in Mice

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin and streptomycin, was adjusted to a concentration of5×10⁷ cells/mL with PBS to prepare a cell suspension. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 7 weeks of ages (CAnN.Cg-Foxn1nu/CrlCrlj,female, Charles River Laboratories Japan, Inc.). Ten days after cellinoculation, a test compound in saline with 100 μM ofhydroxypropyl-β-cyclodextrin was intravenously administered at dosesfrom ½ MTD to MTD. The experiment was conducted on groups eachconsisting of 3 mice. Five days after administration, tumor samples werecollected and fixed with IHC Zinc Fixative (BD Pharmingen) at 4° C. for24 hr. Paraffin-embedded tissues were sectioned (3 μm), mounted onpositively charged slides, and air-dried. Immunohistochemical stainingof CD31 was conducted using Ventana autostainer model Discover XT (RocheDiagnostics) according to the manufacture's protocol. Sections weredeparaffinized, conditioned and the antigens were retrieved with CC1(Ventana Medical Systems). Slides were blocked with Blocker A andBlocker B (Endogenous biotin blocking kit, Roche Diagnostics). Ratanti-mouse IgG CD31 antibody (Dianova GmbH) was applied at 2 μg/mL.Sections were incubated with the antibody for 6 hr, followed by 32minutes incubation with biotinylated anti-rat IgG antibody (JacksonImmunoResearch Laboratories) at 2.2 μg/mL. The detection was performedwith Streptavidin-HRP D for 16 min, followed by incubation with DAB Dand DAB H₂O₂ D (DABMap kit, Ventana Medical Systems, Inc) for 8 min.Slides were counterstained with Hematoxylin II (Roche Diagnostics) for16 min, followed by incubation with Bluing reagent for 4 min. Sectionswere dehydrated in graded ethanols, defatted in xylene replacement andcovered with DPX (Merck KGaA).

Immunostained slides were scanned using Vectra 2 Automated Slide ImagingSystem (Perkin Elmer Inc.). The number of blood vessels in the wholetumor was quantified by counting the CD31-positive objects using inForm2 software (PerkinElmer Inc.) Area of the tumor region was measured byassessing the hematoxylin-staining area using inform 2 software(PerkinElmer Inc.) The number of blood vessels was normalized by thearea of the tumor region. An increase rate of the blood vessel number ofthe test compound-dosing group was calculated with the below formula,and shown in Table 9.Increase rate of blood vessel number(%)=((blood vessel number of thetest compound-dosing group−blood vessel number of the controlgroup)/blood vessel number of the control group)×100

TABLE 9 Increase rate of Test compound Dose (mg/kg) blood vessel number(%) Halichondrin B 0.025 31 0.05 39 Compound (1) 0.10 69 0.20 154

Pharmacological Test Example 10. Effect on α-SMA Positive-CAFs in theFaDu Subcutaneous Model

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin and streptomycin, was adjusted to a concentration of5×10⁷ cells/mL with PBS to prepare a cell suspension. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 5 to 6 weeks of ages(CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Ten days after cell inoculation, a test compound in saline with100 μM of hydroxypropyl-β-cyclodextrin was intravenously administered at½ MTD and MTD. The experiment was conducted on groups each consisting of3 mice. Two days after administration, tumor samples were collected andfixed with IHC Zinc Fixative (BD Pharmingen) at 4° C. for 24 hr.Paraffin-embedded tissues were sectioned (3 μm), mounted on positivelycharged slides, and air-dried for 6 hr. Immunohistochemical staining ofα-SMA was conducted using Ventana autostainer model Discover XT (RocheDiagnostics). Sections were deparaffinized, conditioned and the antigenswere retrieved with proprietary buffers, EZPrep and CC1 (Ventana MedicalSystems). Mouse anti-α-SMA monoclonal antibody conjugated with alkalinephosphatase (clone 1A4, Sigma) was applied at 5 μg/mL. Sections wereincubated with the antibody for 6 hr. The detection was performed withRedMap kit (Ventana Medical Systems, Inc). Sections were dehydrated ingraded ethanols, defatted in xylene replacement and covered with DPX(Merck KGaA). The serial tumor slices were deparaffinized and stainedwith Mayer's hematoxylin (Muto Pure Chemicals) for 1 min. Sections weredehydrated in graded ethanols, defatted in xylene replacement andcovered with DPX (Merck KGaA).

Immunostained slides were scanned using Vectra 2 Automated Slide ImagingSystem (Perkin Elmer Inc.). The area of the α-SMA-positive region in thewhole tumor was quantified by counting the α-SMA-positive objects usinginForm 2 software (PerkinElmer Inc.). Area of the tumor region wasmeasured by assessing the hematoxylin-staining area using inForm 2software (PerkinElmer Inc.). The area of the α-SMA positive region wasnormalized by the area of the tumor region. A suppression rate of theα-SMA positive area of the test compound-dosing group was calculatedwith the below formula, and shown in Table 10.

TABLE 10 Suppression rate of Test compound Dose (mg/kg) α-SMA positivearea (%) Halichondrin B 0.025 7 0.05 3 Compound (1) 0.10 21 0.20 28Suppression rate of α-SMA positive area (%) = − ((α-SMA positive area ofthe test compound-dosing group − α-SMA positive area of the controlgroup)/α-SMA positive area of the control group) × 100

Pharmacological Test Example 11. HSC-2 Orthotopic Transplantation MouseModel

Luciferase-transduced HSC-2-Luc cells were established byretrovirus-mediated gene transfer. First, the DNA fragment encodingfirefly luciferase was obtained from pGL3-enhancer plasmid (GenBank #:U47297), and subcloned into the retroviral vector pCX4pur (GenBank #:AB086386). Then, helper-free recombinant retroviruses were produced bytransfecting the above retroviral expression vector together with pGPand pE-Ampho plasmids (Takara Bio; Shiga, Japan), into 293T cells (ATCC;Manassas, USA). Next, HSC-2 cells were infected with the recombinantretroviruses, and were cultured for two weeks in the presence ofpuromycin (2 μg/mL). The infected cells were selected from a polyclonalproliferative population of the culture.

Under anesthesia, the human SCCHN cell line, HSC-2-Luc was inoculatedinto tongue of female nude mice (1×10⁶ cells in 50 μL of PBS), 6 weeksof ages (CAnN.Cg-Foxn1nu/CrlCrlj mice; Charles River, Inc.; Shizuoka,Japan). Seven days after transplantation, the tumor volume was analyzedusing bioluminescence signal from HSC-2-Luc cells. For bioluminescenceimaging, 0.1 mL of 15 mg/mL D-luciferin (Promega, Madison, Wis.) wasinjected intraperitoneally into nude mice under 1% to 2% inhaledisoflurane anesthesia. The bioluminescence signal was monitored usingthe IVIS SPECTRUM series (PerkinElmer, Waltham, Mass.), consisting of ahighly sensitive, cooled charge coupled device camera. Living Imagesoftware (PerkinElmer, Waltham, Mass.) was used to grid the imaging dataand integrate the total bioluminescence signal in eachregion-of-interest (ROI). All bioluminescence images were acquired witha 1 second exposure. Data were analyzed using total photon flux emission(photons/second) in the ROIs.

On the basis of the total photon flux emission obtained on the first dayof administration, the mice were grouped such that averages of the totalphoton flux emission were substantially equal among the groups. Compound(1) or cisplatin was intravenously administered with or withoutcetuximab (Erbitux, Merck Serono Co., Ltd.) once a week for 3 weeks(Q7D×3 schedule). Two experiments were conducted using the identicalprocedure and all data were collected from the experiments. Each groupconsisted of 16 mice.

The imaging data showed that only the treatment of Compound (1) withcetuximab clearly reduced the bioluminescence signal in all mice afterDay 14 (FIG. 6A-6B). Median survival time (MST) was calculated for eachgroup of treatment as the median of the days of death. Increase LifeSpan (ILS) was calculated by the following formula: ILS (%)=(MST ofanimals treated with test compound−MST of control animals)/MST ofcontrol animals×100. ILS (%) of each test compound is shown in Table 11.

TABLE 11 Dose Cetuximab Test compound (mg/kg) (mg/kg) ILS (%) — — 5 231Cisplatin 5 — 0 5 5 150 Compound (1) 0.09 — 238 0.09 5 >1150

Pharmacological Test Example 12. FaDu s.c. Xenograft Model inCombination with Radiation

Luciferase-transduced FaDu-Luc cells were established byretrovirus-mediated gene transfer. First, the DNA fragment encodingfirefly luciferase was obtained from pGL3-enhancer plasmid (GenBank #:U47297), and subcloned into the retroviral vector pCX4pur (GenBank #:AB086386). Then, helper-free recombinant retroviruses were produced bytransfecting the above retroviral expression vector together with pGPand pE-Ampho plasmids (Takara Bio; Shiga, Japan), into 293T cells (ATCC;Manassas, USA). Next, FaDu cells were infected with the recombinantretroviruses, and were cultured for two weeks in the presence ofpuromycin (2 μg/mL). The infected cells were selected from a polyclonalproliferative population of the culture.

A luciferase-transduced human SCCHN cell line FaDu-Luc, which had beencultured in an RPMI-1640 medium containing 10% FBS, and penicillin andstreptomycin, was adjusted to a concentration of 5×10⁷ cells/mL withHank's Balanced Salt Solution to prepare a cell suspension. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right thigh of nude mice, 6 weeks of ages (CAnN.Cg-Foxn1nu/CrlCrlj,female, Charles River Laboratories Japan, Inc.). Thirteen days after thecell inoculation, the tumor volume was analyzed using bioluminescencesignal from FaDu-Luc cells. For bioluminescence imaging, 0.1 mL of 15mg/mL D-luciferin (Promega, Madison, Wis.) was injectedintraperitoneally into nude mice under 1% to 2% inhaled isofluraneanesthesia. The bioluminescence signal was monitored using the IVISSPECTRUM series (PerkinElmer, Waltham, Mass.), consisting of a highlysensitive, cooled chargecoupled device camera. Living Image software(PerkinElmer, Waltham, Mass.) was used to grid the imaging data andintegrate the total bioluminescence signal in each region-of-interest(ROI). All bioluminescence images were acquired with a 1 secondexposure. Data were analyzed using total photon flux emission(photons/second) in the ROIs. The total photon flux emission wascalculated in accordance with the following calculation formulae:Relative bioluminescence level=Total photon flux emission(day X)/Totalphoton flux emission(the first day)Tumor Regression(%)=(1−minimum Relative bioluminescence level)×100

On the basis of the total photon flux emission obtained on the first dayof administration, the mice were grouped such that averages of the totalphoton flux emission were substantially equal among the groups. Theexperiment was conducted on groups each consisting of 6 mice. Compound(1) was administrated via tail vein injection on day 1 and 8.Irradiation was performed 18 Gy on day 4 and day 11. Tumor regressionfor Compound (1) is shown in Table 12.

TABLE 12 Dose Test compound (mg/kg) Radiation (Gy) Tumor Regression (%)— — 18 16 Compound (1) 0.09 — 0 0.09 18 97

Pharmacological Test Example 13. Antitumor Activity in CT26 SubcutaneousSyngeneic Model in Combination with Anti-mPD-1 Antibody in Mice

A murine undifferentiated colon carcinoma cell line CT26, which had beencultured in an RPMI-1640 medium containing 10% FBS, and penicillin andstreptomycin, was adjusted to a concentration of 2×10⁷ cells/mL withHank's Balanced Salt Solution to prepare a cell suspension. On day 1,the cell suspension was inoculated in a volume of 100 μL into asubcutaneous part of a right flank of BALB/c mice, 6 weeks of ages(BALB/cAnNCrlCrlj, female, Charles River Laboratories Japan, Inc.). Twodays after the cell inoculation, the mice were randomly divided intofour groups and each group consists of 8 mice. The shortest diameter andthe longest diameter of a tumor in each mouse were measured by using anelectronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation),so as to calculate the volume of the tumor in accordance with thefollowing calculation formula:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2T/C=(mean tumor volume of treated group)/(mean tumor volume of controlgroup)Inhibition of tumor growth(%)=(1−T/C)×100

The test compound was intravenously administered at 0.09 mg/kg on days 3and 11. Anti-mPD-1 antibody (BE0146, Bio X Cell) was intravenouslyadministered at 10 mg/kg on days 3, 7, 11, and 15. Inhibition of tumorgrowth on day 15(%) of each test compound is shown in Table 13.

TABLE 13 Dose Anti-mPD-1 Inhibition of tumor growth Test compound(mg/kg) antibody (mg/kg) on day 15 (%) — — 10 32 Compound (1) 0.09 — 300.09 10 62

Pharmacological Test Example 14. Effect on Tubulin Polymerization InVitro (FIG. 10A)

Tubulin Polymerization Assay kit was purchased from Cytoskeleton, Inc.(Cat. #BK011P). The kit contained 1 bottle of lyophilized tubulinprotein purified from porcine brain, 3 tubes of lyophilized GTP, 2bottles of lyophilized assay buffer, and 1 bottle of tubulin glycerolbuffer. Assay buffer was prepared by dissolving the contents in 10 mL ofdeionized and sterilized water. This solution contained 80 mmol/Lpiperazine-N,N′-bis[2-ethanesulfonic acid] sesquisodium salt, 2.0 mmol/Lmagnesium chloride, 0.5 mmol/L ethylene glycol-bis(2-amino-ethyl ether)N,N,N′,N′-tetra-acetic acid, pH 6.9, and 10 μmol/L fluorescent reporter.The buffer was stored at −70° C. until use. Tubulin glycerol bufferconsisted of 80 mmol/L piperazine-N,N′-bis[2-ethanesulfonic acid]sesquisodium salt, 2.0 mmol/L magnesium chloride, 0.5 mmol/L ethyleneglycol-bis(2-amino-ethyl ether) N,N,N′,N′-tetra-acetic acid, and 60% v/vglycerol, pH 6.9. It was stored at 4° C. until use. GTP stock solutionwas prepared by dissolving the contents of each tube in 100 μL ofdeionized and sterilized water to achieve a concentration of 100 mmol/LGTP. Aliquots of this stock were stored at −70° C. until use. Tubulinstock solution (10 mg/mL) was prepared by dissolving the tubulin powderadding 1.1 mL of the mixture of assay buffer and GTP stock solution(100:1, v/v). Aliquots were frozen in liquid nitrogen, and then storedat −70° C. until use.

In the tubulin polymerization assay, reaction mixture was prepared bymixing 820 μL of assay buffer, 17.6 μL of GTP stock solution, and 600 μLof tubulin glycerol buffer. Reaction mixture (1015 μL) was combined with240 μL of the tubulin stock solution. This solution was called astubulin reaction mixture and used for the measurement of test andcontrol wells. No tubulin reaction mixture was prepared by mixing 89.85μL of reaction mixture and 21.25 μL of assay buffer for the measurementof blank wells. The Compound (1) solution (6.25-100 μmol/L; finalconcentrations 0.625-10 μmol/L), or vehicle was added at 5 μL toindividual wells of a 96-well half-area microtiter plate. Tubulinreaction mixture or no tubulin reaction mixture was added at 45 μL toeach well of the plate. Fluorescence emission at 460 nm (excitationwavelength at 360 nm) was measured every 2 minutes for 90 minutes usingthe SpectraMax® M5e microplate reader (Molecular Devices). Tubulinpolymerization was followed by fluorescence enhancement due to theincorporation of a fluorescence reporter into microtubules aspolymerization occurred. The assay was performed in duplicate. The assaydemonstrated that Compound (1) inhibited tubulin polymerization in aconcentration-dependent manner. The fluorescence intensity in each timepoint was calculated by the following formulas:Fluorescence intensity=mean fluorescence measurement of test wells orcontrol wells−mean fluorescence measurement of blank wells;blank well: with vehicle without tubulin; control well: with vehicle andtubulin; test well: with compounds and tubulin.

Pharmacological Test Example 15. Cell-Based Microtubule Dynamics Assay(FIG. 10B)

A cell-based microtubule (MT) dynamics assay was conducted with theU2OS-EB3-AG osteosarcoma cell line, in which the fusion protein of EB3(a microtubule plus end binding protein) and Azami-Green (EB3-AG) wasstably expressed. U2OS-EB3-AG cells were culture in RPMI-1640 mediumcontaining 10% FBS, and penicillin-streptomycin, at 37° C. in ahumidified 5% CO₂ atmosphere. The MT dynamics in the live cells can bevisualized as the movement of the comet-like structure of EB3-AG.U2OS-EB3-AG cells plated on glass-base culture plates (EZVIEW plate, AGCTechno Glass, Japan) were treated with Compound (1) at the indicatedconcentration and the microtubule dynamics were monitored by time-lapseimaging using fluorescent microscope with 60-fold magnificationoil-immersed objective lens (BZ-X710, KEYENCE, Japan). The still imagesat the indicated time points were presented in FIG. 10B. Highermagnification views of the boxed areas were shown in inlets. Whentreated with Compound (1) at 0.5 nM (IC₅₀ value for antiproliferativeactivity in U2OS-EB3-AG cells), the comet-like structures became hard toobserve at around 60 minutes after the addition of the compound. Theseresults clearly demonstrated that Compound (1) had the ability tosuppress the MT dynamics.

Pharmacological Test Example 16. In Vitro Antiproliferative Activity(FIG. 11)

The in vitro antiproliferative assays for Compound (1) were conductedusing a small panel of cancer cell lines including human squamous cellcarcinoma of the esophagus (OE21, TE-8), human adenocarcinoma of theesophagus (OE33), and human uterine sarcoma (MES-SA, MES-SA-Dx5-Rx1).All cell lines were cultured in RPMI-1640 medium containing 10% FBS, andpenicillin-streptomycin (culture medium), in a 5% CO₂ incubator (37°C.). To each well of a 96 well plate (Becton, Dickinson and Company,353219), 75 μL of cell suspension adjusted to a concentration of 4×10⁴cells/mL with the culture medium was added, and the cells were incubatedovernight in a 5% CO₂ incubator (37° C.). On the next day, 25 μL ofCompound (1) in three-fold dilution series suspended in the culturemedium was added to each well, and the resultant was incubated for 72hours in a 5% CO₂ incubator (37° C.). Then, cell viability wasdetermined by CellTiter-Glo® Luminescent Cell Viability Assay (Promega)with 2013 EnVision™ Multilabel Reader (Perkin-Elmer, Wellesley, Mass.).Value of the wells containing cells without adding the test compoundswas defined as 100% and the value of the wells containing no cells wasdefined as 0%. The concentration of Compound (1) necessary forinhibiting the cell growth by 50% (i.e., an IC₅₀ value) was calculate,and is shown in FIG. 11. The P-gp susceptibility was calculated as theratio of the IC₅₀ value in MES-SA-Dx5-Rx1 cells, which overexpress P-gp,to the IC₅₀ value in MES-SA cells.

Pharmacological Test Example 17. Antitumor Effects in the KPL-4Xenograft Models in Mice as Monotherapy; Antitumor Effects in theCOLO-704 Xenograft Models in Mice as Monotherapy (FIG. 12)

A human HER-2 positive breast cancer cell line KPL-4, which had beencultured in a DMEM containing 10% FBS, and penicillin-streptomycin, wasadjusted to a concentration of 1×10⁸ cells/mL with Hanks' Balanced SaltSolution to prepare a cell suspension. The cell suspension wasinoculated in a volume of 100 μL into a subcutaneous part of a rightflank of nude mice, 8 weeks of ages (CAnN.Cg-Foxn1nu/CrlCrlj, female,Charles River Laboratories Japan Inc.). Eleven days after cellinoculation (Day 1), the shortest diameter and the longest diameter of atumor in each mouse were measured by using an electronic digital caliper(Digimatic™ caliper, Mitutoyo Corporation), so as to calculate thevolume of the tumor in accordance with the following calculationformulae:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)Relative body weight(RBW)=Body weight(day X)/Body weight(the first day)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof six mice. The test compound was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 20 μg/kg, 60 μg/kg, or 180μg/kg for 2 weeks (on Day 1 and Day 8). The tumor regression wasobserved in 60 μg/kg- and 180 g/kg-treated groups, and theadministration at 180 μg/kg completely eradicated the xenograft tumorsin all mice on Day 15.

A human ovarian cancer cell line COLO-704, which had been cultured in aRPMI-1640 containing 10% FBS, and penicillin-streptomycin, was adjustedto a concentration of 1×10⁸ cells/mL with Hanks' Balanced Salt Solutionto prepare a cell suspension. The cell suspension was inoculated in avolume of 100 μL into a subcutaneous part of a right flank of nude mice,5 weeks of ages (CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles RiverLaboratories Japan Inc.). Nine days after cell inoculation (Day 1), theshortest diameter and the longest diameter of a tumor in each mouse weremeasured by using an electronic digital caliper (Digimatic™ caliper,Mitutoyo Corporation), so as to calculate the volume of the tumor inaccordance with the following calculation formulae:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter (mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)Relative body weight(RBW)=Body weight(day X)/Body weight(the first day)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof six mice. The test compound was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 20 μg/kg, 60 μg/kg, or 180μg/kg for 2 weeks (on Day 1 and Day 8). The compound treatment inducedtumor regression at 180 μg/kg and tumor growth delay at 60 μg/kg. Theadministration at 180 μg/kg completely eradicated the xenograft tumorsin all mice on Day 22.

Pharmacological Test Example 18. Effect on CD31-Positive Vessel in theFaDu Subcutaneous Model in Mice (FIG. 13)

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin-streptomycin (culture medium), was adjusted to aconcentration of 5×10⁷ cells/mL with culture medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 6 weeks of ages(CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Ten days after cell inoculation, the mice were grouped such thataverages of the tumor volumes were substantially equal among the groups.The experiment was conducted on groups each consisting of 6 mice. Eachtest compound was dissolved in DMSO and a solution was stored in thefreezer before use. Immediately before the administration, the stocksolution was diluted with saline. The test compound in saline wasintravenously administered at 20 μg/kg, 60 μg/kg, or 180 μg/kg. Fivedays after the single administration, tumor samples were collected andfixed with IHC Zinc Fixative (BD Pharmingen) at 4° C. for 24 hours.Paraffin-embedded tissues were sectioned (3 μm), mounted on positivelycharged slides, and air-dried. Immunohistochemical staining of CD31 wasconducted using Ventana autostainer model Discover XT (RocheDiagnostics) according to the manufacture's protocol. Sections weredeparaffinized, conditioned and the antigens were retrieved with CC1(Ventana Medical Systems). Slides were blocked with Blocker A andBlocker B (Endogenous biotin blocking kit, Roche Diagnostics). Ratanti-mouse IgG CD31 antibody (Dianova GmbH) was applied at 2 μg/mL.Sections were incubated with the antibody for 6 hours, followed by 32minutes incubation with biotinylated anti-rat IgG antibody (JacksonImmunoResearch Laboratories) at 2.2 μg/mL. The detection was performedwith Streptavidin-HRP D for 16 minutes, followed by incubation with DABD and DAB H₂O₂ D (DABMap kit, Ventana Medical Systems, Inc.) for 8minutes. Slides were counterstained with Hematoxylin II (RocheDiagnostics) for 16 min, followed by incubation with Bluing reagent for4 minutes. Sections were dehydrated in graded ethanols, defatted inxylene replacement and covered with DPX® (Merck KGaA). Immunostainedslides were scanned using Vectra® 2 Automated Slide Imaging System(Perkin Elmer Inc.). The number of blood vessels in the whole tumor wasquantified by counting the CD31-positive objects using inform 2 software(PerkinElmer Inc.) Area of the tumor region was measured by assessingthe hematoxylin-staining area using inform 2 software (PerkinElmer Inc.)The number of blood vessels was normalized by the area of the tumorregion. The single administration of test compound at doses of 20, 60,and 180 μg/kg increased the tumor blood vessel number. The ratios ofblood vessel number in the test compound-dosing groups compared tonon-treat group were calculated with the below formula:Tumor vessel ratio=blood vessel number of the test compound-dosinggroup/blood vessel number of the non-treat group)

Pharmacological Test Example 19. Effect on α-SMA-Positive CAFs in theFaDu Subcutaneous Model in Mice (FIG. 14)

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin-streptomycin (culture medium), was adjusted to aconcentration of 5×10⁷ cells/mL with culture medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 6 weeks of ages(CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Ten days after cell inoculation, the mice were grouped such thataverages of the tumor volumes were substantially equal among the groups.The experiment was conducted on groups each consisting of 5 mice. Eachtest compound was dissolved in DMSO and a solution was stored in thefreezer before use. Immediately before the administration, the stocksolution was diluted with saline. The test compound in saline wasintravenously administered at 20 μg/kg, 60 μg/kg, or 180 μg/kg. Two daysor 5 days after the single administration, tumor samples were collectedand fixed with IHC Zinc Fixative (BD Pharmingen) at 4° C. for 24 hours.Paraffin-embedded tissues were sectioned (3 μm), mounted on positivelycharged slides, and air-dried. Sections were deparaffinized, conditionedand the antigens were retrieved using microwave with 1 mM EDTA at pH6.0. Sections were blocked with 1% of BSA in TBS. Mouse anti-α-SMAmonoclonal antibody conjugated with alkaline phosphatase (clone 1A4,Sigma) was applied at 5 μg/mL. Sections were incubated with the antibodyfor 2.5 hr. The detection was performed with Fast red II substrate kit(Nichirei Bioscience Inc.). Sections were counterstained with Mayer'sHematoxylin (Muto Pure Chemicals) for 50 seconds. Sections weredehydrated in graded ethanols, defatted in xylene replacement andcovered with DPX (Merck KGaA). Immunostained slides were scanned usingVectra 2 Automated Slide Imaging System (Perkin Elmer Inc.). The area ofα-SMA-positive region in the whole tumor was quantified by counting theα-SMA-positive objects using inform 2 software (PerkinElmer Inc.) Areaof the tumor region was measured by assessing the hematoxylin-stainingarea using inform 2 software (PerkinElmer Inc.). The area of theα-SMA-positive region was normalized by the area of the tumor region.The single administration of test compound significantly reduced theα-SMA positive area at doses of 60 and 180 μg/kg on Day 3 and at a doseof 180 μg/kg on Day 6. A suppression rate of the α-SMA-positive area ofthe test compound-dosing group was calculated with the below formula:α-SMA ratio=α-SMA area of the test compound-dosing group/α-SMA area ofthe non-treat group

Pharmacological Test Example 20. Effects on Tenascin-C andEDA-Fibronectin in the FaDu Subcutaneous Model in Mice (FIG. 15)

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin-streptomycin (culture medium), was adjusted to aconcentration of 5×10⁷ cells/mL with culture medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 6 weeks of ages(CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Ten days after cell inoculation, the mice were grouped such thataverages of the tumor volumes were substantially equal among the groups.The experiment was conducted on groups each consisting of 5 mice.Compound (1) was dissolved in DMSO and a solution was stored in thefreezer before use. Compound (1) (180 μg/kg) and Cetuximab (CTX,Erbitux®, Merck Serono Co. Ltd.) (10 mg/kg) was diluted with saline andintravenously injected on Day 1. Five days after the singleadministration, tumor samples were collected and fixed with IHC ZincFixative (BD Pharmingen) at 4° C. for 24 hr. Paraffin-embedded tissueswere sectioned (3 μm), mounted on positively charged slides, andair-dried. Sections were deparaffinized, conditioned and the antigenswere retrieved using microwave with 1 mM EDTA at pH 6.0 for Tenascin-C.For EDA-fibronectin, the antigens retrieval procedure was not necessary.Sections were incubated with BLOXALL Blocking Solution (Vector Labs) for10 min to block endogenous peroxidase, and with Mouse on Mouse IgBlocking Reagent (Vector Labs) for 1 hour, and then with 2.5% normalhorse serum for 30 minutes. For immunohistochemical staining ofTenascin-C, mouse anti-Tenascin-C monoclonal antibody (clone 4C8MS, IBL)was applied at 5 μg/mL. Sections were incubated with the antibodyovernight at 4° C. For immunohistochemical staining of EDA-fibronectin,mouse anti-EDA-fibronectin monoclonal antibody (clone IST-9, Abcam) wasapplied at 1.5 μg/mL. Sections were incubated with the antibody for 1hour at room temperature. The detection was performed with Mouse OnMouse ImmPRESS™ Peroxidase Polymer Kit (Vector Labs). Sections werecounterstained with Mayer's Hematoxylin (Muto Pure Chemicals) for 50sec. Sections were dehydrated in graded ethanols, defatted in xylenereplacement and covered with DPX (Merck KGaA). Immunostained slides werescanned using Vectra 2 Automated Slide Imaging System (Perkin ElmerInc.). The expression levels of both Tenascin-C and ED-A fibronectinwere reduced in the Compound (1) and CTX treated tumors compared withcontrol tumors.

Pharmacological Test Example 21. Antitumor Effects in FaDu SubcutaneousXenograft Model in Combination with Cetuximab in Mice (FIG. 16)

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin-streptomycin, was adjusted to a concentration of 5×10⁷cells/mL with Hanks' Balanced Salt Solution to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of athymic mice(CAnN.Cg-Foxn1nu/CrlCrlj, female, 7 weeks old, Charles River JapanInc.). Ten days after cell inoculation (Day 1), the length and the widthof a tumor in each mouse were measured by using an electronic digitalcaliper (Mitutoyo Corporation), so as to calculate the volume of thetumor in accordance with the following calculation formula:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)

On the basis of TV, the mice were randomly grouped (Day 1). Each groupwas consisted in six mice. Compound (1) was dissolved in DMSO and asolution was stored in the freezer before use. Compound (1) (20, 60, or180 μg/kg) and Cetuximab (CTX, Erbitux®, Merck Serono Co., Ltd.) (10mg/kg) was diluted with saline and intravenously injected on Day 1.Changes of RTV of each group were shown in FIG. 16. At doses of 180μg/kg and 60 μg/kg, antitumor efficacies of Compound (1) with CTX werestronger than that of CTX monotherapy with tumor regression. Theantitumor efficacy of Compound (1) at doses of 20 mg/kg in combinationwith CTX tended to be stronger than that of CTX monotherapy.

Pharmacological Test Example 22. Antitumor Effects in the Soft TissueSarcoma Xenograft Models in Mice as Monotherapy (FIG. 17)

MES-SA

A human uterine sarcoma cell line MES-SA, which had been cultured in anRPMI-1640 containing 10% FBS, and penicillin-streptomycin, was adjustedto a concentration of 2×10⁸ cells/mL with Hanks' Balanced Salt Solutionto prepare a cell suspension, and the suspension was mixed with Geltrex®(Thermo Fisher Scientific Inc., #A1413202) in a ratio of 1:1 to preparea cell suspension in a concentration of 1×10⁸ cells/mL. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 6 weeks of ages (CAnN.Cg-Foxn1nu/CrlCrlj,female, Charles River Laboratories Japan Inc.). Six days after cellinoculation (Day 1), the shortest diameter and the longest diameter of atumor in each mouse were measured by using an electronic digital caliper(Digimatic™ caliper, Mitutoyo Corporation), so as to calculate thevolume of the tumor in accordance with the following calculationformulae:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof 6 mice. The test compound was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 180 μg/kg for 2 weeks (onDay 1 and Day 8). The antitumor activity was observed with tumor growthdelay in the treated group.

HT-1080

A human fibrosarcoma cell line HT-1080, which had been cultured in anE-MEM containing 10% FBS, NEAA and antibiotics was adjusted to aconcentration of 3×10⁷ cells/mL with medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of athymic mice(CAnN.Cg-Foxn1nu/CrlCrlj, female, 6 weeks old, Charles River JapanInc.). Six days after cell inoculation (Day 1), the length and the widthof a tumor in each mouse were measured by using an electronic digitalcaliper (Mitutoyo Corporation), so as to calculate the volume of thetumor in accordance with the following calculation formula:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)

On the basis of TV, the mice were randomly grouped (Day 1). Each groupwas consisted in six mice. Compound (1) was dissolved in DMSO and asolution was stored in the freezer before use. Compound (1) (180 μg/kg)was diluted with saline and intravenously injected on Day 1 and Day 8.Changes of RTV of each group was shown in FIG. 17. The antitumoractivity was observed with tumor regression in the treated group.

CTG-2041

Tumor fragments of human angiosarcoma CTG-2041 were implanted s.c. inthe left flank of female mice. Tumor growth was monitored twice a weekusing digital caliper, so as to calculate the volume of the tumor inaccordance with the following calculation formula:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)

When the volume of tumors reached approximately 200 mm³, animals arematched by tumor volume into treatment or control groups and dosinginitiated on Day 1. Each group was consisted with five mice. Compound(1) was dissolved in DMSO and a solution was stored in the freezerbefore use. Compound (1) (100 μg/kg) diluted in saline and intravenouslyinjected on Day 1 and Day 8. Changes of RTV of each group were shown inFIG. 17. The antitumor activity was observed with tumor regression inthe treated group.

Pharmacological Test Example 23. Antitumor Effects in the EndometrialCancer Sarcoma Xenograft Models in Mice as Monotherapy (FIG. 18)

HEC-108

A human endometrial cancer cell line HEC-108, which had been cultured inan E-MEM containing 15% FBS and antibiotics were adjusted to aconcentration of 7.14×10⁷ cells/mL with medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 150 μLinto a subcutaneous part of a right flank of athymic mice(CAnN.Cg-Foxn1nu/CrlCrlj, female, 6 weeks old, Charles River JapanInc.). Thirteen days after cell inoculation (Day 1), the length and thewidth of a tumor in each mouse were measured by using an electronicdigital caliper (Mitutoyo Corporation), so as to calculate the volume ofthe tumor in accordance with the following calculation formula:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)

On the basis of TV, the mice were randomly grouped (Day 1). Each groupwas consisted in six mice. Compound (1) was dissolved in DMSO and asolution was stored in the freezer before use. Compound (1) (180 μg/kg)was diluted in saline and intravenously injected on Day 1 and Day 8.Changes of RTV of each group was shown in FIG. 18. The antitumoractivity was observed with tumor growth delay in the treated group.

AN3CA

A human endometrial cancer cell line AN3CA, which had been cultured inan E-MEM containing 10% FBS, and penicillin-streptomycin, was adjustedto a concentration of 1.4×10⁸ cells/mL with Hanks' Balanced SaltSolution to prepare a cell suspension, and the suspension was mixed withGeltrex® (Thermo Fisher Scientific Inc., #A1413202) in a ratio of 1:1 toprepare a cell suspension in a concentration of 7×10⁷ cells/mL. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 6 weeks of ages (CAnN.Cg-Foxn1nu/CrlCrlj,female, Charles River Laboratories Japan Inc.). Twelve days after cellinoculation (Day 1), the shortest diameter and the longest diameter of atumor in each mouse were measured by using an electronic digital caliper(Digimatic™ caliper, Mitutoyo Corporation), so as to calculate thevolume of the tumor in accordance with the following calculationformulae:Tumor volume(mm³)=Longest diameter(mm)×Shortest diameter(mm)×Shortestdiameter(mm)/2Relative tumor volume(RTV)=Tumor volume(day X)/Tumor volume(the firstday)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof five mice. The test compound was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 180 μg/kg for 2 weeks (onDay 1 and Day 8). The antitumor activity was observed with tumorregression in the treated group.

Pharmacological Test Example 24. NCI-N87 and MKN-28 Growth InhibitionAssay

In this assay, the growth inhibitory activities of test compounds inhuman gastric cancer cell lines NCI-N87 and MKN-28 were measured,respectively. NCI-N87 and MKN-28 cells were maintained in RPMI-1640medium containing 10% FBS, penicillin and streptomycin in a 5% CO₂incubator (37° C.). To each well of a 96-well plate (Becton, Dickinsonand Company, 353219), 100 μL of NCI-N87 or MKN-28 cell suspensionadjusted to a concentration of 3×10⁴ cells/mL with the culture mediumwas added, and the cells were incubated overnight in a 5% CO₂ incubator(37° C.). On the next day, 100 μL of Compound (1) or Halichondrin B inthree-fold dilution series suspended in the culture medium was added toeach well, and the resultant was incubated for 3 days in a 5% CO₂incubator (37° C.). Then, cell viability was determined byCellTiter-Glo® Luminescent Cell Viability Assay (Promega) with EnVision2103 Multilabel Reader (Perkin-Elmer, Wellesley, Mass.). Value of thewells containing cells without adding the test compounds was defined as100% and the value of the wells containing no cells was defined as 0%.The concentrations of the test compound necessary for inhibiting thecell growth by 50% (i.e., an IC₅₀ value) were calculated and are shownin Table 14.

TABLE 14 NCI-N87 MKN-28 Test compound (IC₅₀ (nM)) (IC₅₀ (nM))Halichondrin B 0.007 0.017 Compound (1) 0.002 0.015

Pharmacological Test Example 25. HuTu 80 Growth Inhibition Assay

In this assay, the growth inhibitory activities of test compounds in thehuman small bowel cancer cell line HuTu 80, which was isolated fromduodenal tissue were measured. HuTu 80 cells were maintained in EMEMmedium containing 10% FBS, penicillin and streptomycin in a 5% CO₂incubator (37° C.). To each well of a 96-well plate (Becton, Dickinsonand Company, 353219), 100 μL of a HuTu80 cell suspension adjusted to aconcentration of 3×10⁴ cells/mL with the culture medium was added, andthe cells were incubated overnight in a 5% CO₂ incubator (37° C.). Onthe next day, 100 μL of Compound (1) or Halichondrin B in three-folddilution series suspended in the culture medium was added to each well,and the resultant was incubated for 3 days in a 5% CO₂ incubator (37°C.). Then, cell viability was determined by CellTiter-Glo® LuminescentCell Viability Assay (Promega) with EnVision 2103 Multilabel Reader(Perkin-Elmer, Wellesley, Mass.). Value of the wells containing cellswithout adding the test compounds was defined as 100% and the value ofthe wells containing no cells was defined as 0%. The concentrations ofthe test compounds necessary for inhibiting the cell growth by 50%(i.e., an IC₅₀ value) were calculated and are shown in Table 15.

TABLE 15 HuTu 80 Test compound (IC₅₀ (nM)) Halichondrin B 0.031 Compound(1) 0.019

Pharmacological Test Example 26. SW780 Growth Inhibition Assay

In this assay, the growth inhibitory activities of test compounds in thehuman urothelial cancer cell line SW780 were measured. SW780 cells weremaintained in RPMI-1640 medium containing 10% FBS, penicillin, andstreptomycin in a 5% CO₂ incubator (37° C.). To each well of a 96-wellplate (Becton, Dickinson and Company, 353219), 100 μL of a SW780 cellsuspension adjusted to a concentration of 3×10⁴ cells/mL with theculture medium was added, and the cells were incubated overnight in a 5%CO₂ incubator (37° C.). On the next day, 100 μL of Compound (1) orHalichondrin B in three-fold dilution series suspended in the culturemedium was added to each well, and the resultant was incubated for 3days in a 5% CO₂ incubator (37° C.). Then, cell viability was determinedby CellTiter-Glo® Luminescent Cell Viability Assay (Promega) withEnVision 2103 Multilabel Reader (Perkin-Elmer, Wellesley, Mass.). Valueof the wells containing cells without adding the test compounds wasdefined as 100% and the value of the wells containing no cells wasdefined as 0%. The concentrations of the test compounds necessary forinhibiting the cell growth by 50% (i.e., an IC₅₀ value) were calculated,and are shown in Table 16.

TABLE 16 SW780 Test compound (IC₅₀ (nM)) Halichondrin B 0.032 Compound(1) 0.017

Pharmacological Test Example 27. HS-SY-II Growth Inhibition Assay

In this assay, the growth inhibitory activities of test compounds in thehuman synovial sarcoma cell line HS-SY-II were measured. SH-SY-II cellswere maintained in a DMEM medium containing 10% FBS, penicillin, andstreptomycin in a 5% CO₂ incubator (37° C.). To each well of a 96-wellplate (Becton, Dickinson and Company, 353219), 100 μL of a HS-SY-II cellsuspension adjusted to a concentration of 3×10⁴ cells/mL with theculture medium was added, and the cells were incubated overnight in a 5%CO₂ incubator (37° C.). On the next day, 100 μL of Compound (1) orHalichondrin B in three-fold dilution series suspended in the culturemedium was added to each well, and the resultant was incubated for 3days in a 5% CO₂ incubator (37° C.). Then, cell viability was determinedby CellTiter-Glo® Luminescent Cell Viability Assay (Promega) withEnVision 2103 Multilabel Reader (Perkin-Elmer, Wellesley, Mass.). Valueof the wells containing cells without adding the test compounds wasdefined as 100% and the value of the wells containing no cells wasdefined as 0%. The concentrations of the test compounds necessary forinhibiting the cell growth by 50% (i.e., an IC₅₀ value) were calculated,and are shown in Table 17.

TABLE 17 HS-SY-II Test compound (IC₅₀ (nM)) Halichondrin B 0.010Compound (1) 0.002

EQUIVALENTS AND SCOPE

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

What is claimed is:
 1. A compound selected from the group consisting of:

and salts thereof.
 2. The compound of claim 1, wherein the compound isof Formula (D-5), or a salt thereof.
 3. The compound of claim 1, whereinthe compound is of Formula (D-4), or a salt thereof.